JPH07313164A - Method for continuous reaction using biocatalyst - Google Patents
Method for continuous reaction using biocatalystInfo
- Publication number
- JPH07313164A JPH07313164A JP11125194A JP11125194A JPH07313164A JP H07313164 A JPH07313164 A JP H07313164A JP 11125194 A JP11125194 A JP 11125194A JP 11125194 A JP11125194 A JP 11125194A JP H07313164 A JPH07313164 A JP H07313164A
- Authority
- JP
- Japan
- Prior art keywords
- reactor
- reaction
- biocatalyst
- activity
- substrate
- 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
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は酵素、微生物菌体などの
生体触媒に基質を反応させて種々の有用物質を製造する
方法において、反応を効率よく実施するための方法及び
装置に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for efficiently carrying out the reaction in a method for producing various useful substances by reacting a substrate with a biocatalyst such as an enzyme or a microbial cell. .
【０００２】[0002]
【従来の技術】酵素、微生物菌体などの生体触媒は高い
基質特異性を有すること、常温常圧下で効率よく触媒反
応が進むことから、近年種々の有用物質の生産に利用さ
れている。例えば、酵素あるいは酵素活性を有する微生
物菌体を反応の溶媒に不溶の担体に結合あるいは担体で
包括的に包むことあるいは限外ろ過膜などで仕切ること
によって実質的に反応系外に出ないようにすることによ
って生体触媒とし、これを反応器に充填し、ここに基質
液を連続的に供給することによって反応を行い、流出し
た反応液から種々の方法によって生成物を回収すること
が行われている。2. Description of the Related Art Biocatalysts such as enzymes and microbial cells have a high substrate specificity and can be efficiently catalyzed at room temperature and atmospheric pressure, so that they have been utilized for producing various useful substances in recent years. For example, by binding an enzyme or a microbial cell having an enzyme activity to a carrier insoluble in the solvent of the reaction or comprehensively wrapping it with a carrier or partitioning with an ultrafiltration membrane or the like so that it does not substantially go out of the reaction system. As a result, a biocatalyst is obtained, which is filled in a reactor, and a substrate solution is continuously supplied to carry out the reaction, and the product is recovered from the reaction solution flowing out by various methods. There is.
【０００３】しかしながら、生体触媒を用いる反応にお
いては、長期間の反応によって反応の活性が低下する事
によって、反応器から流出した液中の残存基質濃度が上
昇し、製品の純度や、製品コスト等の面から、ある一定
期間反応を行った後、生体触媒の交換を行わざるを得な
いのが現状である。このような活性低下は生体触媒が変
性などの変化をうけやすいタンパク質から構成されてい
る以上避け得ないことではあるが、生体触媒の交換を行
うために連続反応を停止することは、経済的にも負担が
大きい。特に発熱反応などの酵素活性が低下しやすい場
合には、活性低下を避けるために反応熱の除去が行いや
すい特殊な反応器を使用するなどの設備的な工夫が行わ
れるが、このような場合でも生体触媒の交換もより頻繁
に行わなければならない。このような方式で連続反応を
行うと設備的な負担あるいは人件費の負担が大きくなっ
てしまう。However, in a reaction using a biocatalyst, the activity of the reaction is lowered by the reaction for a long period of time, so that the concentration of the residual substrate in the liquid flowing out from the reactor is increased, and the purity of the product, the product cost, etc. In view of this, the present situation is that the biocatalyst must be replaced after the reaction for a certain period of time. Such a decrease in activity is unavoidable because the biocatalyst is composed of proteins that are susceptible to changes such as denaturation, but it is economically economical to stop the continuous reaction in order to exchange the biocatalyst. Is also a heavy burden. Especially when the enzyme activity such as exothermic reaction is likely to decrease, in order to avoid the activity decrease, equipment such as using a special reactor that is easy to remove reaction heat is used. However, the biocatalyst must be replaced more frequently. If the continuous reaction is carried out by such a method, the burden of equipment or labor cost will increase.
【０００４】このような連続形式で有用物質を生産する
方法の一つとして、特開昭６０−８７７８３では固定化
増殖微生物を用いる連続醗酵方法として、生産性の低下
した最終段のリアクタの固定化増殖微生物を先頭段に持
ってくることによって、再活性化しながら連続醗酵する
方法が提案されている。この方法では栄養分の不足、生
産物による阻害などで生産性が低下したリアクタを先頭
段にもってくることによって新鮮培地が供給されるよう
にし、それによって低下した生産性が回復できるとして
いる。しかしながら基質濃度が非常に高い生体触媒反
応、又は酵素もしくは増殖能を失った菌体を用いる反応
系においては、特開昭６０−８７７８３で提案されてい
るような新鮮培地での再活性化などを行うことはできな
い。As one of the methods for producing a useful substance in such a continuous mode, Japanese Patent Laid-Open No. 60-87783 discloses a continuous fermentation method using immobilized growing microorganisms to immobilize a reactor at the final stage with reduced productivity. A method has been proposed in which continuous fermentation is carried out while reactivating by bringing a proliferating microorganism to the first stage. According to this method, a fresh medium is supplied by bringing a reactor whose productivity has been lowered due to lack of nutrients, inhibition by products, etc., to recover the lowered productivity. However, in a biocatalytic reaction with a very high substrate concentration, or in a reaction system using an enzyme or a bacterial cell that has lost the ability to grow, reactivation in a fresh medium as proposed in JP-A-60-87783 is required. I can't do it.
【０００５】[0005]
【発明が解決しようとする課題】従って本発明は、酵素
系が本来増殖能を有しない場合、又は酵素系をその増殖
を許容しない条件下で用いる場合においても、長時間に
わたり安定に連続的に酵素反応を行うことができる方法
を提供しようとするものである。Therefore, the present invention provides a stable and continuous method for a long period of time even when the enzyme system originally does not have a growth ability or when the enzyme system is used under conditions that do not allow its growth. It is intended to provide a method capable of performing an enzymatic reaction.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、連続生体
触媒反応において酵素反応学的な見地から、鋭意検討し
た結果、本発明を完成するに至った。すなわち、本発明
は酵素あるいは酵素活性含有物などの生体触媒が充填さ
れている、少なくとも３個以上の反応器を直列に連結し
た装置を用いて連続生体触媒反応を行う際、各反応器の
反応性の低下に応じて、最も反応性の低下した反応器の
連結状態を変更し、残りの２個以上の反応器で連続反応
を行いながら、反応性の低下した反応器の生体触媒を新
鮮な生体触媒と交換し、交換後、反応器の連結の最後段
になるように連結することを特徴とする生体触媒を用い
た連続反応方法に関するものである。Means for Solving the Problems The present inventors have completed the present invention as a result of intensive studies from the viewpoint of enzymatic reaction in a continuous biocatalytic reaction. That is, according to the present invention, when a continuous biocatalytic reaction is carried out using an apparatus in which at least three or more reactors are filled with a biocatalyst such as an enzyme or an enzyme-active substance, the reaction of each reactor is performed. Depending on the decrease in reactivity, the connection state of the reactor with the lowest reactivity is changed, and the biocatalyst of the reactor with the reduced reactivity is made fresh while performing continuous reaction in the remaining two or more reactors. The present invention relates to a continuous reaction method using a biocatalyst, which is characterized in that the biocatalyst is replaced with a biocatalyst, and after the replacement, the biocatalyst is connected to the last stage of the connection.
【０００７】従って本発明は、生体触媒が充填され且つ
３個以上直列に連結される反応器を用いて連続的酵素反
応により基質を有用物質に転換する方法において、
（１）前記直列に連結された反応器の内最上流の反応器
に基質を含有する反応媒体を導入し、そして最下流の反
応器から有用物質を含有する反応済媒体を取り出し、
（２）前記直列に連結された反応器の内酵素活性が最も
低下した生体触媒を収容する反応器を反応系から切り離
し、該生体触媒を新鮮な生体触媒と交換し、次に（３）
上記（２）において新鮮な生体触媒を充填した反応器を
反応系に最下流の反応器として導入し、そして（４）前
記（１）〜（３）の工程を複数回反復する、ことを特徴
とする方法を提供する。Therefore, the present invention provides a method of converting a substrate into a useful substance by a continuous enzymatic reaction using a reactor filled with biocatalysts and connected in series of three or more,
(1) A reaction medium containing a substrate is introduced into the most upstream reactor of the reactors connected in series, and a reacted medium containing a useful substance is taken out from the most downstream reactor,
(2) The reactor containing the biocatalyst having the lowest enzyme activity in the reactors connected in series is disconnected from the reaction system, and the biocatalyst is replaced with a fresh biocatalyst, and then (3).
The reactor filled with fresh biocatalyst in (2) above is introduced into the reaction system as the most downstream reactor, and (4) the above steps (1) to (3) are repeated a plurality of times. And provide a method.
【０００８】通常、前記（２）における酵素活性が最も
低下した生体触媒を収容する反応器が、前記（１）にお
ける最上流の反応器である。本発明の方法は、酵素反応
が発熱反応である場合に特に有用である。例えば、アス
パルターゼまたはアスパルターゼ活性含有物を用いたフ
マル酸からのＬ−アスパラギン酸の製造反応や、酒石酸
エポキシターゼまたは酒石酸エポキシターゼ活性含有物
を用いたシスエポキシコハク酸からのＬ−酒石酸の製造
反応などがあげられる。Usually, the reactor containing the biocatalyst having the lowest enzyme activity in the above (2) is the most upstream reactor in the above (1). The method of the present invention is particularly useful when the enzymatic reaction is an exothermic reaction. For example, a production reaction of L-aspartic acid from fumaric acid using aspartase or an aspartase activity-containing substance, and a production of L-tartaric acid from cis-epoxysuccinic acid using a tartaric acid epoxidase or a tartaric acid epoxidase-containing substance Examples include reactions.
【０００９】[0009]
【具体的な説明】本発明において、「生体触媒」という
場合、反応に関与する酵素及び菌体のみならず、菌体破
砕物、種々の程度に精製した酵素調製物、例えば硫酸ア
ンモニウム塩析により分別した酵素調製物、アセトン等
の有機溶媒沈澱により分別した酵素調製物等、種々の酵
素活性含有物さらには上記の酵素及び／又は酵素活性含
有物を種々の担体に固定化したものも含まれる。[Detailed Description] In the present invention, when referring to “biocatalyst”, not only the enzyme and the cells involved in the reaction but also the crushed cells, enzyme preparations purified to various extents, for example, fractionation by ammonium sulfate salting out The enzyme preparations described above, the enzyme preparations separated by precipitation with an organic solvent such as acetone, and the like, and various enzyme activity-containing substances, and the above-mentioned enzymes and / or enzyme activity-containing substances immobilized on various carriers are also included.
【００１０】本発明は、生体触媒がそれ自体増殖能を有
しないものである場合、例えば酵素や部分精製された酵
素や菌体破砕物を使用する場合、あるいは菌体はそれ自
体増殖能を有しているが実際には増殖しえない、又は死
滅してしまう様な条件下で使用する場合に特に有用であ
る。本発明で扱う生体触媒反応では、通常は基質濃度が
高いため、微生物は実質的に死滅しており、一般の醗酵
での生産物阻害や、代謝老廃物などによる生産性の低下
などの問題はないが、その一方、微生物が死滅している
ため反応に必要な酵素は種々の要因によってその活性を
失い、反応器の反応性が低下する。このような酵素の失
活の要因の中で、大きな影響を与えるものとして反応温
度があげられる。酵素はタンパク質で構成されており、
反応に必要な構造を維持するためには一定以下の温度で
反応を行う必要がある。In the present invention, when the biocatalyst does not have the ability to grow itself, for example, when an enzyme, a partially purified enzyme or a crushed product of the cells is used, or the cells have the ability to grow themselves. However, it is particularly useful when used under conditions in which it does not actually grow or is killed. In the biocatalytic reaction treated in the present invention, since the substrate concentration is usually high, the microorganisms are substantially dead, and there are problems such as product inhibition in general fermentation and decrease in productivity due to metabolic waste products. However, on the other hand, since the microorganisms are dead, the enzyme required for the reaction loses its activity due to various factors, and the reactivity of the reactor decreases. Among such factors of enzyme deactivation, the reaction temperature has a great influence. Enzymes are composed of proteins,
In order to maintain the structure required for the reaction, it is necessary to carry out the reaction at a temperature below a certain temperature.
【００１１】しかしながら、反応が発熱反応の場合に
は、一定温度を保つために冷却などの操作が行われる
が、生体触媒を反応器に充填して用いる場合には、効率
良く冷却するのが非常に困難であり、冷却が十分に行え
ないために反応活性が低下してしまうことがしばしば起
こる。一般的には、反応速度面からは、反応温度が高い
方が反応速度も大きく、生産性が良くなるが、先に述べ
たように反応温度が高いと、生体触媒の活性低下も大き
くなるため、生体触媒の交換を頻繁に行わなければなら
なくなる。However, when the reaction is an exothermic reaction, an operation such as cooling is performed to maintain a constant temperature, but when the biocatalyst is filled in the reactor and used, it is very important to cool it efficiently. However, the reaction activity often decreases due to insufficient cooling. Generally, from the viewpoint of reaction rate, the higher the reaction temperature, the higher the reaction rate and the better the productivity, but as described above, the higher the reaction temperature, the greater the decrease in the activity of the biocatalyst. Therefore, the biocatalyst must be replaced frequently.
【００１２】そこで本発明では酵素反応学的な見地から
生体触媒を利用した発熱反応の場合、反応速度の非常に
大きい条件の第１反応器（最上流の反応器）とそれより
も反応速度が小さい第２、第３の反応器等複数の反応器
を用いて連続反応を行う。通常、直列に連結した反応器
で生体触媒を用いて反応を行う場合、各反応器の通液速
度は同じになる。一方、酵素反応的には、基質濃度が低
くなると、反応速度も低下する。Therefore, in the present invention, in the case of an exothermic reaction using a biocatalyst from the viewpoint of enzymatic reaction, the reaction rate is much higher than that of the first reactor (the most upstream reactor) under the condition that the reaction rate is very large. A continuous reaction is carried out using a plurality of reactors such as a small second and third reactor. Usually, when a reaction is carried out using a biocatalyst in reactors connected in series, the liquid passing rate of each reactor is the same. On the other hand, in terms of enzymatic reaction, when the substrate concentration is low, the reaction rate is also low.
【００１３】よって、複数の反応器を直列に連結した場
合、高濃度基質が流入する最上流の反応器の反応速度が
最も大きく、後段になるほど反応速度は小さくなる。す
なわち、発熱反応の場合、最上流の反応器で最も発熱が
大きく、後段になるほど発熱が小さくなる。従って、最
上流の反応器では生体触媒の活性低下も大きくなるが、
基質濃度が高いため、反応速度は比較的大きく保つこと
ができる。第２反応器ではある程度反応が進み、基質濃
度が低下した液が流入する。Therefore, when a plurality of reactors are connected in series, the reaction speed of the uppermost flow reactor into which the high-concentration substrate flows is the highest, and the reaction speed becomes lower toward the latter stage. That is, in the case of an exothermic reaction, the most heat is generated in the most upstream reactor, and the heat generation decreases in the subsequent stages. Therefore, although the activity of the biocatalyst decreases greatly in the most upstream reactor,
Due to the high substrate concentration, the reaction rate can be kept relatively high. In the second reactor, the reaction proceeds to some extent, and the liquid with the lowered substrate concentration flows in.
【００１４】ここでは第１（最上流）反応器ほどの速度
の反応は起こらず、よって発熱も第１反応器ほど大きく
ない。そのため生体触媒の活性低下も第１（最上流）反
応器より小さくなる。しかしながら、基質濃度が低くな
るため、反応を進めるためには、第１（最上流）反応器
よりも反応活性の高い生体触媒が必要となる。同様にし
て、第３反応器ではさらに基質濃度の低い液が流入する
ため、発熱による生体触媒の活性低下はあまりないが、
反応を完結させるためには生体触媒の反応活性としては
第２反応器以上の活性のものが必要となる。Here, the reaction at the rate as high as that of the first (upstream) reactor does not occur, and therefore the heat generation is not so large as that of the first reactor. Therefore, the decrease in the activity of the biocatalyst is smaller than that in the first (upstream flow) reactor. However, since the substrate concentration becomes low, a biocatalyst having higher reaction activity than that of the first (upstream) reactor is required to proceed the reaction. Similarly, since the liquid having a lower substrate concentration flows into the third reactor, the activity of the biocatalyst is not significantly reduced by heat generation,
In order to complete the reaction, the reaction activity of the biocatalyst needs to be higher than that of the second reactor.
【００１５】このような条件を整理してみると、第１反
応器では生体触媒の活性低下が起こっても、基質濃度が
高いため比較的反応速度を大きく保つことができる。ま
た後の反応器ほど基質濃度が低くなるので、反応を完結
させるためには、反応活性の高い生体触媒が必要になる
が、ここでは前の反応器ほど反応速度が大きくないの
で、生体触媒の活性低下は比較的小さくなる。As a summary of such conditions, even if the activity of the biocatalyst is lowered in the first reactor, the reaction rate can be kept relatively high because of the high substrate concentration. Further, since the substrate concentration becomes lower in the later reactor, a biocatalyst with high reaction activity is required to complete the reaction, but here the reaction rate is not as high as in the previous reactor, The decrease in activity is relatively small.
【００１６】以上のような観点で生体触媒を用いた連続
発熱反応を行う方法を提供する。すなわち生体触媒の活
性低下が起きやすい第１（最上流）反応器の反応性が低
下した際に、その生体触媒を反応活性の高いもの（新鮮
なもの）に交換した後、反応を完結させるために必要な
反応器として最終段（最下流）に持っていくことによっ
て、連続反応を停止させることなく、また酵素反応学的
にも理想の状態で反応を行うことができる。From the above viewpoints, a method for carrying out a continuous exothermic reaction using a biocatalyst is provided. That is, when the reactivity of the first (upstreammost) reactor where the activity of the biocatalyst is likely to decrease becomes low, the reaction is completed after the biocatalyst is replaced with one having a high reaction activity (fresh). By bringing it to the final stage (downstream) as a reactor required for the above, the reaction can be performed in an ideal state in terms of enzymatic reaction without stopping the continuous reaction.
【００１７】具体的には、反応とともに活性が低下した
最上流の反応器の生体触媒を活性の高いもの（新鮮なも
の）に交換後、最終段（最下流）に連結することの繰り
返しで、常に活性の最も高い反応器が最終段になってお
り、前段にすすむほど活性が低い反応器になっている。
このような構成では、活性低下を前提として反応速度を
大きくし、反応をある程度進めるための第１（最上流）
反応器と、基質濃度が低くなるため反応速度がそれほど
大きくないが活性低下の小さい、反応を完結させるため
の反応器を組み合わせることによって生体触媒の活性低
下と反応速度の面から、生体触媒を無駄なく、最大限利
用することができるようになる。Specifically, the biocatalyst in the most upstream reactor, which has decreased in activity with the reaction, is replaced with a highly active one (fresh) and then connected to the final stage (most downstream), The reactor with the highest activity is always in the final stage, and the lower the activity is, the lower the reactor is.
In such a configuration, the reaction rate is increased on the assumption that the activity is decreased, and the first (upstream) method for promoting the reaction to some extent
Biocatalyst is wasted from the viewpoint of biocatalyst activity reduction and reaction rate by combining a reactor and a reactor for completing the reaction, which reaction rate is not so large because the substrate concentration is low but the activity decrease is small. Instead, you will be able to get the most out of it.
【００１８】以下に本発明の方法について図面をまじえ
て実施態様を説明するが、本発明はかかる実施態様のみ
に限定されるものではない。生体触媒を充填した３個以
上の反応器を直列に連結した装置を用い、最上流の反応
器に基質液を連続的に供給して有用物質を生産する際、
所定期間後に最上流の反応器の生体触媒を活性の高いも
のに交換後、最下流の反応器になるように反応器相互の
連結状態を変更することによって行う。Embodiments of the method of the present invention will be described below with reference to the drawings, but the present invention is not limited to such embodiments. When using a device in which three or more reactors filled with a biocatalyst are connected in series and a substrate solution is continuously supplied to the most upstream reactor to produce a useful substance,
After a predetermined period of time, the biocatalyst in the most upstream reactor is exchanged with a highly active one, and then the connection state between the reactors is changed so that it becomes the most downstream reactor.
【００１９】このような連続反応方法において、各反応
器に要求される条件として以下のようなことがあげられ
る。まず最上流の反応器では、流入した基質液の大部分
を反応させるように供給速度を調節する。この反応器で
は反応の大部分を行うため発熱量も大きく、よって生体
触媒の活性低下も比較的大きい。そのため所定期間経過
後、生体触媒を交換した後、最後段に接続する。In such a continuous reaction method, the conditions required for each reactor are as follows. First, in the most upstream reactor, the feed rate is adjusted so that most of the inflowing substrate liquid is reacted. Since most of the reaction is carried out in this reactor, the amount of heat generated is large, and therefore the activity of the biocatalyst is also significantly reduced. Therefore, after a predetermined period of time, the biocatalyst is replaced and then connected to the last stage.
【００２０】第２段以下の反応器では基質濃度が低くな
った条件下で反応の転化率を平衡状態にまで高めるため
に第１反応器よりも高い生体触媒活性が必要となる。以
下に図面に基づいて説明する。第１図は多段直列反応装
置の概略図であり、（Ａ），（Ｂ）及び（Ｃ）は反応器
であり、生体触媒が充填されている。これらの反応器に
基質液供給管（１）が開閉弁（４）を介して接続されて
おり、前段の反応器の流出液が順次後段の反応器に供給
管（２）によって開閉弁（５）を介して相互に連結され
ている。また各反応器には反応終了液の抜き出し管
（３）が開閉弁（６）を介して接続されている。In the second and subsequent reactors, higher biocatalytic activity is required than in the first reactor in order to increase the conversion rate of the reaction to an equilibrium state under the condition where the substrate concentration is low. A description will be given below with reference to the drawings. FIG. 1 is a schematic diagram of a multi-stage series reaction apparatus, in which (A), (B) and (C) are reactors, each of which is filled with a biocatalyst. A substrate liquid supply pipe (1) is connected to these reactors via an on-off valve (4), and the effluent of the preceding reactor is sequentially supplied to the latter reactor by an on-off valve (5). ) Are connected to each other. Further, a pipe (3) for withdrawing the reaction completion liquid is connected to each reactor through an on-off valve (6).
【００２１】この装置を用いて、基質液供給管の開閉弁
（４ｂ）及び（４ｃ）を閉じ、反応器相互の供給管の開
閉弁（５ｃ）を閉じ、反応終了液の抜き出し管の開閉弁
（６ａ），（６ｂ）を閉じると、基質液が反応器（Ａ）
に入り反応器（Ｂ）を経て、反応器（Ｃ）から反応終了
液が得られる。このように連結された反応器では、最初
の反応器である（Ａ）では基質濃度が高いため、反応速
度が大きいが、反応にともなう発熱が大きいため、生体
触媒の活性低下も大きくなる。Using this apparatus, the on-off valves (4b) and (4c) of the substrate liquid supply pipe are closed, the on-off valves (5c) of the supply pipes of the reactors are closed, and the on-off valve of the extraction pipe of the reaction completion liquid is closed. When (6a) and (6b) are closed, the substrate solution becomes the reactor (A).
A reaction-terminated liquid is obtained from the reactor (C) via the reactor (B). In the reactor thus connected, the reaction rate is high because the substrate concentration is high in the first reactor (A), but since the heat generation accompanying the reaction is large, the activity of the biocatalyst is also greatly reduced.
【００２２】それに対して反応器（Ｂ）及び（Ｃ）では
順次発熱も小さくなるため生体触媒の活性低下も小さく
なる。一定期間後、反応器（Ａ）の生体触媒の活性が低
下してきたら、供給管の開閉弁（５ａ）を閉じ、基質供
給管の開閉弁（４ａ）を閉じるとともに、開閉弁（４
ｂ）を開ける。そうすると基質液が反応器（Ｂ）に供給
され、反応器（Ｃ）から反応終了液が得られるようにな
る。このようにして連続反応系のラインからはずした反
応器（Ａ）の生体触媒を活性の高い新しいもの（新鮮な
もの）に交換した後、供給管の開閉弁（５ｃ）を開ける
とともに、反応終了液の抜き出し管の開閉弁（６ａ）を
開け、（６ｃ）を閉じる。On the other hand, in the reactors (B) and (C), the heat generation becomes smaller in sequence, and therefore the decrease in the activity of the biocatalyst becomes smaller. When the activity of the biocatalyst in the reactor (A) decreases after a certain period of time, the on-off valve (5a) of the supply pipe is closed, the on-off valve (4a) of the substrate supply pipe is closed, and the on-off valve (4
b) open. Then, the substrate solution is supplied to the reactor (B), and the reaction completed solution can be obtained from the reactor (C). In this way, after replacing the biocatalyst in the reactor (A) removed from the continuous reaction system line with a new one with high activity (fresh), the on-off valve (5c) of the supply pipe is opened and the reaction is completed. The on-off valve (6a) of the liquid withdrawal pipe is opened and (6c) is closed.
【００２３】次に反応器（Ｂ）の反応活性が低下してき
たら、連結状態を変えることによって連続反応系から反
応器（Ｂ）をきりはなし、生体触媒を交換後、反応器
（Ａ）の後に連結することによって、活性の最も高い反
応器が最後段に位置するようにする。以下同様に反応器
（Ｃ）をきりはなし、生体触媒を交換後、反応器（Ｂ）
の後ろに連結する。このように生体触媒の活性が低下し
た反応器を（Ａ），（Ｂ），（Ｃ）の順に連続反応系か
らきりはなし、生体触媒を交換後、連続反応系の下流に
連結する。Next, when the reaction activity of the reactor (B) begins to decrease, the connection state is changed to disconnect the reactor (B) from the continuous reaction system, replace the biocatalyst, and after the reactor (A). By linking, the reactor with the highest activity is positioned at the last stage. Similarly, after removing the reactor (C) and replacing the biocatalyst, the reactor (B)
Connect behind. In this way, the reactor in which the activity of the biocatalyst is lowered is removed from the continuous reaction system in the order of (A), (B), and (C), and after the biocatalyst is exchanged, it is connected to the downstream of the continuous reaction system.
【００２４】このように連結を変え、最上流の反応器の
生体触媒を交換後、最下流に連結することによって、連
続反応を停止せずに生体触媒の交換を行い、活性の高い
反応器（新鮮な生体触媒）を常に最後段に位置させる。
そうすることによって、生体触媒の活性低下、反応速度
の両面からその触媒機能を十分に活用できるようにな
る。By changing the connection in this way and exchanging the biocatalyst of the most upstream reactor, the biocatalyst is exchanged without stopping the continuous reaction by connecting the biocatalyst at the most downstream side, so that the reactor with high activity ( Always place the fresh biocatalyst at the end.
By doing so, it becomes possible to fully utilize the catalytic function of the biocatalyst in terms of both activity reduction and reaction rate.
【００２５】本発明において使用できる生体触媒は、連
続反応が行えるものであればいずれでも使用できる。例
えば酵素や酵素活性含有物を種々の担体に固定化したも
の、また酵素活性含有物を固定化せずに、用いることも
できる。例えば、反応器を工夫する事によって実質的に
生体触媒が反応器の外に漏れでないようにすることによ
って、反応器中にとどまるようにすればよい。好ましい
固定化担体としてはイオン交換樹脂、セルロース等の吸
着体、寒天ゲル、カラギーナン、アルギン酸ゲル、ポリ
アクリルアミドゲル、ポリビニルアルコールゲル、カゼ
インゲル、マンナンゲルなどの包括体等があげられる。
これらの担体に酵素を吸着、あるいは酵素や微生物菌体
を包括的に固定化する事によって固定化生体触媒を調製
することができる。As the biocatalyst which can be used in the present invention, any biocatalyst can be used as long as it can carry out a continuous reaction. For example, the enzyme or the enzyme activity-containing substance immobilized on various carriers, or the enzyme activity-containing substance without being immobilized can be used. For example, by devising the reactor, the biocatalyst does not substantially leak out of the reactor so that it stays in the reactor. Examples of preferable immobilization carriers include ion exchange resins, adsorbents of cellulose and the like, inclusion bodies such as agar gel, carrageenan, alginic acid gel, polyacrylamide gel, polyvinyl alcohol gel, casein gel and mannan gel.
An immobilized biocatalyst can be prepared by adsorbing an enzyme on these carriers or by comprehensively immobilizing the enzyme or microbial cells.
【００２６】これらのなかでも、イオン交換樹脂やカラ
ギーナン、寒天ゲル、マンナンゲルなどが特に好適であ
る。また、酵素活性含有物をそのまま用いる場合には、
反応器の出口に市販の限外濾過膜モジュールなどを装備
し、反応器から抜きだした液が限外濾過モジュールを通
った後、酵素活性含有物は反応器に循環され、流出液が
次の反応器に供給されるようにすればよい。Among these, ion exchange resins, carrageenan, agar gel, mannan gel and the like are particularly preferable. Further, when using the enzyme activity-containing substance as it is,
The outlet of the reactor is equipped with a commercially available ultrafiltration membrane module, etc., and after the liquid extracted from the reactor passes through the ultrafiltration module, the enzyme activity content is circulated to the reactor and the effluent is It may be supplied to the reactor.
【００２７】また本発明に使用できる反応器は生体触媒
が実質的に漏れ出ない形式のものであればとくに限定さ
れないが、ジャケットや冷却コイル、外部循環冷却装置
などの冷却装置が装備されているか、または反応器全体
を恒温槽中に入れるなどの方法によって、冷却が行える
ようにしたものを用いるほうが望ましい。また反応器の
間に熱交換器を挿入することも可能である。The reactor that can be used in the present invention is not particularly limited as long as the biocatalyst does not substantially leak out, but is it equipped with a cooling device such as a jacket, a cooling coil, and an external circulation cooling device? Alternatively, it is preferable to use a reactor which can be cooled by putting the whole reactor in a constant temperature bath. It is also possible to insert a heat exchanger between the reactors.
【００２８】[0028]
【実施例】次に本発明の方法を実施例をあげて説明する
が、本発明はかかる実施例のみに限定されるものではな
い。実施例１． ２Ｌジャーファーメンターにフマル酸２０
ｇ、リン酸１カリウム１ｇ、硫酸マグネシウム７水塩
０．５ｇ、酵母エキス２０ｇ、コーンスティープリカー
２０ｇを水に溶解し、pHをアンモニアで６．８に調節し
た培地１Ｌを仕込み滅菌した後、別に５００ml振盪フラ
スコに同上の培地５０mlをいれて培養しておいたＥｓｃ
ｈｅｒｉｃｈｉａ ｃｏｌｉ ＡＴＣＣ １１３０３を
接種し、３７℃で通気攪拌培養した。培地中有のフマル
酸が消失した時点で培養液に酢酸を加え、pHを５に調節
し、４５℃で１時間放置後、培養液を遠心分離にかけ、
菌体を分離した。EXAMPLES The method of the present invention will now be described with reference to examples, but the present invention is not limited to these examples. Example 1. Fumaric acid 20 in 2L jar fermenter
g, 1 potassium phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, yeast extract 20 g, corn steep liquor 20 g were dissolved in water and charged with 1 L of a medium whose pH was adjusted to 6.8 with ammonia, followed by sterilization Esc cultivated by adding 50 ml of the same medium to a 500 ml shake flask
herichia coli ATCC 11303 was inoculated and cultured at 37 ° C. with aeration and stirring. When the fumaric acid in the medium disappeared, acetic acid was added to the culture solution to adjust the pH to 5, and the culture solution was centrifuged at 45 ° C. for 1 hour.
The cells were separated.
【００２９】この菌体を４０℃の３％カラギーナン水溶
液１００mlに加えて混合し、２％塩化カリウム水溶液５
００ml中にシリンジから滴下して直径約４mmの球状ゲル
とした。このゲルを固定化生体触媒として用い、その４
０mlを図−１に示す３個の反応器（直径３cm、長さ８c
m）それぞれに充填し、反応器全体を３５℃の恒温器に
入れ、１Ｌ中にフマル酸２００ｇ、硫酸マグネシウム７
水塩０．２ｇを含有するフマル酸アンモニウム水溶液
（pH８．３）を２４ml／hrの速度で流通させて連続反応
を行った。連続反応を開始するにあたって基質液が反応
器（Ａ）から（Ｂ），（Ｃ）に流通するように開閉弁を
セットしておいた。The cells were added to 100 ml of a 3% carrageenan aqueous solution at 40 ° C. and mixed, and a 2% potassium chloride aqueous solution was added.
A spherical gel having a diameter of about 4 mm was dropped from a syringe into 00 ml. Using this gel as an immobilized biocatalyst, part 4
3 ml (diameter 3 cm, length 8 c
m) Fill each of them, put the whole reactor in a thermostat of 35 ° C., and put 200 g of fumaric acid and 7 g of magnesium sulfate in 1 L.
An ammonium fumarate aqueous solution (pH 8.3) containing 0.2 g of a water salt was circulated at a rate of 24 ml / hr to carry out a continuous reaction. The on-off valve was set so that the substrate solution could flow from the reactor (A) to (B) and (C) when starting the continuous reaction.
【００３０】反応開始時には反応器（Ａ）の流出液中の
残存フマル酸濃度は４％であり最終反応器（Ｃ）の流出
液中のフマル酸濃度は０．１８％であった。反応開始か
ら１４日目に反応器（Ａ）の流出液の残存フマル酸濃度
が６％に上昇していた。このとき最終反応器（Ｃ）の流
出液中の残存フマル酸濃度は０．１８％であった。さら
に反応を継続し反応開始から３０日目には反応器（Ａ）
の流出液中のフマル酸濃度が１０％にまで上昇し、最終
反応器（Ｃ）の流出液中の残存フマル酸濃度は０．３５
％に上昇していた。At the start of the reaction, the residual fumaric acid concentration in the effluent of the reactor (A) was 4%, and the fumaric acid concentration in the effluent of the final reactor (C) was 0.18%. On the 14th day from the start of the reaction, the residual fumaric acid concentration in the effluent of the reactor (A) had increased to 6%. At this time, the residual fumaric acid concentration in the effluent of the final reactor (C) was 0.18%. The reaction is further continued, and the reactor (A) is operated on the 30th day from the start of the reaction.
The fumaric acid concentration in the effluent of the product was increased to 10%, and the residual fumaric acid concentration in the effluent of the final reactor (C) was 0.35.
% Had risen.
【００３１】この時点で開閉弁を操作して反応器相互の
連結状態を変え、反応器（Ａ）を連続反応系からきりは
なすとともに、反応器（Ａ）の固定化生体触媒を新しい
ものと交換し、反応器（Ｃ）の後ろに位置するように開
閉弁を操作して連結状態を変えた。この操作で基質液は
反応器（Ｂ）から（Ｃ），（Ａ）を通って通液されるよ
うになった。この状態で連続反応を継続し、連結状態を
変えてから２０日目に反応器（Ｂ）を連続系からきりは
なし、生体触媒を交換すると共に、反応器（Ａ）の後ろ
に位置するように再度連結した。このようにして、２０
日ごとに先頭の反応器の生体触媒を新しいものと交換し
ながら連続反応を続けた結果、６ケ月にわたって転換率
９８．５モル％以上で連続反応を行うことができ、残存
フマル酸濃度の上昇もみられなかった。At this time, the on-off valve is operated to change the connection state of the reactors, the reactor (A) is separated from the continuous reaction system, and the immobilized biocatalyst of the reactor (A) is replaced with a new one. Then, the connection state was changed by operating the on-off valve so that it was located behind the reactor (C). By this operation, the substrate liquid came to pass from the reactor (B) through (C) and (A). Continue the continuous reaction in this state, and remove the reactor (B) from the continuous system on the 20th day after changing the connection state, replace the biocatalyst, and place it behind the reactor (A). Reconnected. In this way, 20
As a result of continuing the continuous reaction while replacing the biocatalyst in the first reactor with a new one every day, the continuous reaction could be carried out at a conversion rate of 98.5 mol% or more for 6 months, and the residual fumaric acid concentration increased. I couldn't see it.
【００３２】比較例１．実施例１と同様の方法で固定化
生体触媒を調製し、同様に３個の反応器に充填し、反応
器全体を３７℃の恒温器に入れ、１Ｌ中にフマル酸２０
０ｇ、硫酸マグネシウム７水塩０．２ｇを含有するフマ
ル酸アンモニウム水溶液（pH８．３）を２４ml／hrの速
度で流通させる連続反応を行った。連続反応を開始する
にあたって基質液が反応器（Ａ）から（Ｂ），（Ｃ）に
流通するように開閉弁をセットしておいた。反応開始時
には反応器（Ａ）の流出液中の残存フマル酸濃度は４％
であり最終反応器（Ｃ）の流出液中のフマル酸濃度は
０．１８％であった。反応開始から３０日目に反応器
（Ａ）の流出液の残存フマル酸濃度が１０％に上昇して
いた。このとき最終反応器（Ｃ）の流出液中の残存フマ
ル酸濃度は０．３％に上昇していた。 Comparative Example 1. An immobilized biocatalyst was prepared in the same manner as in Example 1, similarly charged in three reactors, and the whole reactor was put in a 37 ° C. incubator and 20 L of fumaric acid was added to 1 L.
A continuous reaction was carried out by flowing an ammonium fumarate aqueous solution (pH 8.3) containing 0 g and 0.2 g of magnesium sulfate heptahydrate at a rate of 24 ml / hr. The on-off valve was set so that the substrate solution could flow from the reactor (A) to (B) and (C) when starting the continuous reaction. At the start of the reaction, the residual fumaric acid concentration in the effluent of the reactor (A) was 4%.
The fumaric acid concentration in the effluent of the final reactor (C) was 0.18%. On the 30th day from the start of the reaction, the concentration of residual fumaric acid in the effluent of the reactor (A) had risen to 10%. At this time, the residual fumaric acid concentration in the effluent of the final reactor (C) had risen to 0.3%.
【００３３】この時点で開閉弁を操作して反応器相互の
連結状態を変え、反応器（Ｃ）を連続反応系からきりは
なすとともに、反応器（Ｃ）の固定化生体触媒を新しい
ものと交換し、反応器（Ａ）の前に位置するように開閉
弁を操作して連結状態を変えた。この操作で基質液は反
応器（Ｃ）から（Ａ），（Ｂ）を通って通液されるよう
になった。この状態で連続反応を継続したが、連結状態
を変えてから７日目に反応器（Ｂ）からの流出液中のフ
マル酸濃度が０．３９％となった。そこで反応器（Ｂ）
を連続系からきりはなし、生体触媒を交換すると共に、
反応器（Ｃ）の前に位置するように再度連結した。この
ようにして、連続運転を継続した。流出液中のフマル酸
濃度を０．３％以下に保って連続反応するためには５日
ごとに固定化生体触媒の交換を行わなければならなかっ
た。At this point, the on-off valve is operated to change the connected state of the reactors, so that the reactor (C) is separated from the continuous reaction system and the immobilized biocatalyst in the reactor (C) is replaced with a new one. Then, the connection state was changed by operating the on-off valve so as to be positioned in front of the reactor (A). By this operation, the substrate solution came to flow from the reactor (C) through (A) and (B). The continuous reaction was continued in this state, but the fumaric acid concentration in the effluent from the reactor (B) reached 0.39% on the 7th day after the connection state was changed. So reactor (B)
From the continuous system, replace the biocatalyst,
It was reconnected so that it was located in front of the reactor (C). In this way, continuous operation was continued. The immobilized biocatalyst had to be replaced every 5 days in order to carry out continuous reaction while keeping the fumaric acid concentration in the effluent at 0.3% or less.
【００３４】実施例２．アシネトバクター タルタロゲ
ネス（Ａｃｉｎｅｔｏｂａｃｔｅｒ ｔａｒｔａｒｏｇ
ｅｎｅｓ ＡＴＣＣ ３１１０５）を１Ｌ当たり、シス
エポキシコハク酸２ナトリウム５ｇ、硫酸アンモニウム
３ｇ、リン酸１カリウム１．５ｇ、リン酸２ナトリウム
１．５ｇ、硫酸マグネシウム・７水塩０．５ｇ、硫酸鉄
・７水塩１０mg、塩化カルシウム・２水塩１０mg、硫酸
マンガン・４水塩２０mg、酵母エキス０．１ｇを含有す
る液体培地（pH６．２）１００mlに接種し、３０℃、２
４時間振盪培養し、これを上記と同組成の培地３Ｌを仕
込んだ５Ｌジャーファーメンターに接種して３０℃で通
気攪拌培養を行った。培地中の有機酸濃度が０．０１％
以下に低下した時点で培養液を遠心分離し、菌体を分離
した。 Example 2. Acinetobacter tartarogenesu (Acinetobacter tartarog
enes ATCC 31105) per liter, 5 g of disodium cis-epoxysuccinate, 3 g of ammonium sulfate, 1.5 g of potassium phosphate, 1.5 g of disodium phosphate, 0.5 g of magnesium sulfate-7-hydrate, iron sulfate-7-water Inoculate 100 ml of a liquid medium (pH 6.2) containing 10 mg of salt, 10 mg of calcium chloride / dihydrate, 20 mg of manganese sulfate / tetrahydrate, and 0.1 g of yeast extract at 30 ° C, 2
After shaking culture for 4 hours, this was inoculated into a 5 L jar fermenter charged with 3 L of the medium having the same composition as the above, and aeration stirring culture was carried out at 30 ° C. Organic acid concentration in the medium is 0.01%
The culture was centrifuged at the time when it decreased to below to separate the bacterial cells.
【００３５】この菌体を４０℃の３％カルギーナン水溶
液１００mlに加えて混合し、２％塩化カリウム水溶液５
００ml中にシリンジから滴下して直径約４mmの球状ゲル
とした。このゲル４０mlを固定化生体触媒として図−１
に示す３個の反応器（直径３cm、長さ８cm）に充填し、
反応器全体を２８℃の恒温器に入れ、１Ｌ中にシスエポ
キシコハク酸を２００ｇを含有するシスエポキシコハク
酸２ナトリウム水溶液（pH８．０）を２０ml／hrの速度
で流通させて連続反応を行った。This bacterial cell was added to 100 ml of a 3% carrageenan aqueous solution at 40 ° C. and mixed, and a 2% potassium chloride aqueous solution 5
A spherical gel having a diameter of about 4 mm was dropped from a syringe into 00 ml. 40 ml of this gel is used as an immobilized biocatalyst.
Fill 3 reactors (diameter 3 cm, length 8 cm) shown in
The entire reactor was placed in a thermostat at 28 ° C, and a disodium cis-epoxysuccinate aqueous solution (pH 8.0) containing 200 g of cis-epoxysuccinic acid in 1 L was circulated at a rate of 20 ml / hr for continuous reaction. It was
【００３６】連続反応を開始するにあたって基質液が反
応器（Ａ）から（Ｂ），（Ｃ）に流通するように開閉弁
をセットしておいた。反応開始時には反応器（Ａ）の流
出液中の残存シスエポキシコハク酸濃度は５％であり最
終反応器（Ｃ）の流出液中のシスエポキシコハク酸濃度
は０．１％以下であった。反応開始から７日目に反応器
（Ａ）の流出液の残存シスエポキシコハク酸濃度が７％
に上昇していた。このとき最終反応器（Ｃ）の流出液中
の残存シスエポキシコハク酸濃度は０．２％であった。The opening / closing valve was set so that the substrate solution could flow from the reactors (A) to (B) and (C) when starting the continuous reaction. At the start of the reaction, the residual cis-epoxysuccinic acid concentration in the effluent of the reactor (A) was 5%, and the cis-epoxysuccinic acid concentration in the effluent of the final reactor (C) was 0.1% or less. 7 days after the start of the reaction, the residual cis-epoxysuccinic acid concentration in the effluent of the reactor (A) was 7%.
Was rising to. At this time, the residual cis-epoxysuccinic acid concentration in the effluent of the final reactor (C) was 0.2%.
【００３７】さらに反応を継続し反応開始から１４日目
には反応器（Ａ）の流出液中のシスエポキシコハク酸濃
度が１２％にまで上昇し、最終反応器（Ｃ）の流出液中
の残存シスエポキシコハク酸濃度は０．４％に上昇して
いた。この時点で開閉弁を操作して反応器相互の連結状
態を変え、反応器（Ａ）を連続反応系からきりはなすと
ともに、反応器（Ａ）の固定化生体触媒を新しいものと
交換し、反応器（Ｃ）の後ろに位置するように開閉弁を
操作して連結状態を変えた。この操作で基質液は反応器
（Ｂ）から（Ｃ），（Ａ）を通って通液されるようにな
った。The reaction was further continued, and on the 14th day from the start of the reaction, the concentration of cis-epoxysuccinic acid in the effluent of the reactor (A) increased to 12%, and the effluent of the final reactor (C) contained the effluent. The residual cis-epoxysuccinic acid concentration had risen to 0.4%. At this time, the on-off valve is operated to change the connection state between the reactors, the reactor (A) is separated from the continuous reaction system, and the immobilized biocatalyst in the reactor (A) is replaced with a new one to carry out the reaction. The connection state was changed by operating the on-off valve so that it was located behind the container (C). By this operation, the substrate liquid came to pass from the reactor (B) through (C) and (A).
【００３８】この状態で連続反応を継続し、連結状態を
変えてから１４日目に反応器（Ｂ）を連続系からきりは
なし、固定化生体触媒を交換すると共に、反応器（Ａ）
の後ろに位置するように再度連結した。このようにし
て、１４日ごとに先頭の反応器の固定化生体触媒を新し
いものと交換しながら連続反応を続けた結果、以後３ケ
月にわたって転換率９７．５モル％以上で連続反応を行
うことができ、残存シスエポキシコハク酸濃度は常に
０．５％以下であった。Continuous reaction is continued in this state, and 14 days after changing the connection state, the reactor (B) is disconnected from the continuous system, the immobilized biocatalyst is replaced, and the reactor (A) is removed.
It was connected again so that it would be located behind. In this way, the continuous reaction was continued every 14 days while replacing the immobilized biocatalyst in the first reactor with a new one, and as a result, the continuous reaction was performed at a conversion rate of 97.5 mol% or more for the next 3 months. The residual cis-epoxysuccinic acid concentration was always 0.5% or less.
【図１】図１は本発明の方法を実施するための装置の一
例を示す。1 shows an example of a device for carrying out the method of the invention.
Ａ，Ｂ，Ｃ…反応器 １…基質液供給管 ２…反応器間供給管 ３…反応終了液抜き出し管 ４…基質供給管用開閉弁 ５…反応器間開閉弁 ６…反応終了液抜き出し管用開閉弁 A, B, C ... Reactor 1 ... Substrate liquid supply pipe 2 ... Inter-reactor supply pipe 3 ... Reaction completion liquid extraction pipe 4 ... Substrate supply pipe opening / closing valve 5 ... Inter-reactor opening / closing valve 6 ... Reaction completion liquid extraction pipe opening / closing valve
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:01） (Ｃ１２Ｐ 13/20 Ｃ１２Ｒ 1:19） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI technical display area C12R 1:01) (C12P 13/20 C12R 1:19)
Claims (6)
連結される反応器を用いて連続的酵素反応により基質を
有用物質に転換する方法において、 （１）前記直列に連結された反応器の内最上流の反応器
に基質を含有する反応媒体を導入し、そして最下流の反
応器から有用物質を含有する反応済媒体を取り出し、 （２）前記直列に連結された反応器の内酵素活性が最も
低下した生体触媒を収容する反応器を反応系から切り離
し、該生体触媒を新鮮な生体触媒と交換し、次に （３）上記（２）において新鮮な生体触媒を充填した反
応器を反応系に最下流の反応器として導入し、そして （４）前記（１）〜（３）の工程を複数回反復する、 ことを特徴とする方法。1. A method for converting a substrate into a useful substance by a continuous enzymatic reaction using a reactor in which three or more biocatalysts are connected in series, (1) the reactors connected in series The reaction medium containing the substrate is introduced into the most upstream reactor of the above, and the reacted medium containing the useful substance is taken out from the most downstream reactor, and (2) the enzyme in the reactor connected in series. The reactor containing the biocatalyst having the lowest activity is separated from the reaction system, the biocatalyst is replaced with a fresh biocatalyst, and then (3) the reactor filled with the fresh biocatalyst in (2) above. Introduced into the reaction system as the most downstream reactor, and (4) repeating the steps (1) to (3) a plurality of times.
した生体触媒を収容する反応器が、前記（１）における
最上流の反応器である、請求項１に記載の方法。2. The method according to claim 1, wherein the reactor containing the biocatalyst having the lowest enzyme activity in (2) is the most upstream reactor in (1).
２に記載の方法。3. A biocatalyst is immobilized and used.
The method described in 2.
３に記載の方法。4. The method according to claim 1, wherein the reaction is an exothermic reaction.
The method according to 3.
ルターゼ活性含有物を用いたフマル酸からのＬ−アスパ
ラギン酸の製造反応である請求項１〜４に記載の方法。5. The method according to claim 1, wherein the reaction is a production reaction of L-aspartic acid from fumaric acid using aspartase or a substance containing aspartase activity.
酒石酸エポキシターゼ活性含有物を用いたシスエポキシ
コハク酸からのＬ−酒石酸の製造反応である請求項１〜
４に記載の方法。6. The reaction according to claim 1, which is a reaction for producing L-tartaric acid from cis-epoxysuccinic acid using a tartaric acid epoxidase or a substance containing a tartaric acid epoxidase activity.
The method according to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11125194A JP3635106B2 (en) | 1994-05-25 | 1994-05-25 | Biocatalytic continuous reaction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11125194A JP3635106B2 (en) | 1994-05-25 | 1994-05-25 | Biocatalytic continuous reaction method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07313164A true JPH07313164A (en) | 1995-12-05 |
| JP3635106B2 JP3635106B2 (en) | 2005-04-06 |
Family
ID=14556444
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11125194A Expired - Fee Related JP3635106B2 (en) | 1994-05-25 | 1994-05-25 | Biocatalytic continuous reaction method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3635106B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080138867A1 (en) * | 2006-12-06 | 2008-06-12 | Dayton Christopher L G | Continuous Process and Apparatus for Enzymatic Treatment of Lipids |
| CN111549089A (en) * | 2020-04-23 | 2020-08-18 | 山东立海润生物技术有限公司 | Method for producing 7-aminocephalosporanic acid by enzyme method |
-
1994
- 1994-05-25 JP JP11125194A patent/JP3635106B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080138867A1 (en) * | 2006-12-06 | 2008-06-12 | Dayton Christopher L G | Continuous Process and Apparatus for Enzymatic Treatment of Lipids |
| US8361763B2 (en) * | 2006-12-06 | 2013-01-29 | Bunge Oils, Inc. | Continuous process and apparatus for enzymatic treatment of lipids |
| EP2094824B1 (en) * | 2006-12-06 | 2013-07-03 | Bunge Oils, Inc | A continuous process and apparatus for enzymatic treatment of lipids |
| CN111549089A (en) * | 2020-04-23 | 2020-08-18 | 山东立海润生物技术有限公司 | Method for producing 7-aminocephalosporanic acid by enzyme method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3635106B2 (en) | 2005-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cooney | Bioreactors: design and operation | |
| Fukui et al. | Immobilized microbial cells | |
| US7432091B2 (en) | Highly efficient hydrogen production method using microorganism | |
| CN109153966B (en) | Biotechnological process for producing acrylamide and related novel strain | |
| CN107208085A (en) | Immobilized cell and preparation method thereof | |
| Chibata et al. | Immobilized microbial cells and their applications | |
| CN207828318U (en) | A kind of continuous synthesis system of non-natural amino acid | |
| CN109097409A (en) | The preparation method of D- amino acid and alpha keto acid | |
| JP3635106B2 (en) | Biocatalytic continuous reaction method | |
| JPS62289A (en) | Enzymatic production of l-alpha-amino acid from alpha-ketoic acid | |
| Chibata et al. | Immobilized cells: Historical background | |
| CA1210719A (en) | Method of immobilizing enzymes | |
| JP2677602B2 (en) | Method for producing L-sorbose by passage seed culture and apparatus used therefor | |
| JPS62111693A (en) | Production of l-amino acid from alpha-ketocarboxilic acid byfermentation | |
| Chibata et al. | [43] Continuous l-alanine production using two different immobilized microbial cell preparations on an industrial scale | |
| JPH06277081A (en) | Production of 5-aminolevulinic acid by methane-producing bacterium | |
| JPH07313163A (en) | Method for continuous reaction using biocatalyst | |
| CN218666019U (en) | A breeding device for microorganism immobilization | |
| Cooney | Bioreactors: Design and operation | |
| Chibata et al. | Immobilized cells | |
| JPS5840093A (en) | Production of dextrorotatory lactic acid by means of sporogenous bacterium | |
| BIOCATALYSTS | INDUSTRIAL PRODUCTION OF OPTICALLY ACTIVE | |
| JP2006006344A (en) | Method for producing organic acid | |
| JP3233878B2 (en) | Method for producing L-aspartic acid | |
| JPS615789A (en) | Process for reaction by immobilized biocatalyst |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040824 |
|
| A521 | Written amendment |
Effective date: 20041007 Free format text: JAPANESE INTERMEDIATE CODE: A523 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20041130 |
|
| A61 | First payment of annual fees (during grant procedure) |
Effective date: 20041228 Free format text: JAPANESE INTERMEDIATE CODE: A61 |
|
| R150 | Certificate of patent (=grant) or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |