JPH0793877B2 - Two-step reaction method - Google Patents
Two-step reaction methodInfo
- Publication number
- JPH0793877B2 JPH0793877B2 JP11247789A JP11247789A JPH0793877B2 JP H0793877 B2 JPH0793877 B2 JP H0793877B2 JP 11247789 A JP11247789 A JP 11247789A JP 11247789 A JP11247789 A JP 11247789A JP H0793877 B2 JPH0793877 B2 JP H0793877B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- enzyme
- membrane
- microorganism
- step reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は例えばグルコースを微生物によりエタノールに
変換し、更に酵素の作用により酢酸に変換する場合等に
利用される二段反応法に関するものである。TECHNICAL FIELD The present invention relates to a two-step reaction method used, for example, when glucose is converted into ethanol by a microorganism and further converted into acetic acid by the action of an enzyme. .
（従来の技術） 例えばグルコースを微生物によりエタノールに変換し、
更に酵素の作用により酢酸に変換するような場合には、
二段階の反応を行わせる必要がある。このため従来行わ
れていた方法は次の通りである。(Prior art) For example, glucose is converted into ethanol by a microorganism,
Furthermore, when it is converted to acetic acid by the action of an enzyme,
It is necessary to carry out a two-step reaction. Therefore, the conventional method is as follows.
即ち、第２図に示されるように、まず原液Ａ（グルコー
ス）を微生物又は酵素ｍの入った第１の反応槽（11）に
供給してその内部で中間生成物Ｂ（エタノール）を生成
させる。この反応液を第１の濾過膜（12）にポンプ（1
3）により送って中間生成物Ｂと微生物又は酵素ｍとを
分離し、中間生成物Ｂを第２の反応槽（14）へ供給する
とともに微生物又は酵素ｍを第１の反応槽（11）に戻
す。更に第２の反応槽（14）においては酵素Ｅにより最
終生成物Ｃ（酢酸）を生成させ、これを第２の濾過膜
（15）にポンプ（16）により送って同様に最終生成物Ｃ
と酵素Ｅとを分離し、最終生成物Ｃのみを取り出す方法
である。That is, as shown in FIG. 2, first, the stock solution A (glucose) is supplied to the first reaction tank (11) containing the microorganism or the enzyme m to generate the intermediate product B (ethanol) therein. . The reaction solution is pumped to the first filtration membrane (12) (1
3) to separate the intermediate product B from the microorganism or enzyme m, and to supply the intermediate product B to the second reaction tank (14) and the microorganism or enzyme m to the first reaction tank (11). return. Further, in the second reaction tank (14), the final product C (acetic acid) is produced by the enzyme E, and the final product C (acetic acid) is sent to the second filtration membrane (15) by the pump (16).
And enzyme E are separated, and only the final product C is taken out.
しかしこのような従来の二段反応法は、二つずつの反応
槽と濾過膜とが必要となりコスト高となる欠点があっ
た。However, such a conventional two-step reaction method has a drawback that it requires two reaction tanks and two filtration membranes, resulting in high cost.
（発明が解決しようとする課題） 本発明は上記したような従来の問題点を解決して、二段
反応を単一の反応槽と分離膜のみを用いて行わせること
ができる二段反応法を提供するために完成されたもので
ある。(Problems to be Solved by the Invention) The present invention solves the conventional problems as described above and allows a two-step reaction to be carried out using only a single reaction tank and a separation membrane. Has been completed in order to provide.
（課題を解決するための手段） 上記の課題を解決するためになされた本発明は微生物又
は酵素の作用により第１段の反応を行わせた反応液を表
面と内部の一方又は双方に酵素を固定化した膜体に送
り、この膜体の酵素の作用により第２段の反応を行わせ
ると同時に膜体により反応液中の微生物又は酵素を分離
し、微生物又は酵素を含む不透過液を第１段の反応槽へ
還流させるとともに最終生成物を外部に取り出すことを
特徴とするものである。(Means for Solving the Problems) The present invention made to solve the above problems is to provide a reaction solution in which the reaction of the first stage is carried out by the action of a microorganism or an enzyme with an enzyme on one or both of the surface and the inside. It is sent to the immobilized membrane, and the second step reaction is performed by the action of the enzyme of this membrane, and at the same time, the microorganism or the enzyme in the reaction solution is separated by the membrane, and the impermeable liquid containing the microorganism or the enzyme is separated. It is characterized in that the final product is taken out to the outside while being refluxed to the one-stage reaction tank.
以下に本発明を第１図を参照しつつ更に詳細に説明す
る。Hereinafter, the present invention will be described in more detail with reference to FIG.
第１図において（１）は反応槽、（２）はポンプ、
（３）は分離膜である。反応槽（１）の内部には微生物
又は酵素ｍがあり、原液Ａはこの微生物又は酵素ｍとの
第１段の反応により中間生成物Ｂに変換される。この中
間生成物Ｂと原液Ａと微生物又は酵素ｍとの混じった反
応液は、ポンプ（２）によって分離膜（３）へ送られる
が、本発明においては分離膜（３）として、表面と内部
の一方又は双方に第２段の反応を行わせるための酵素Ｅ
を固定化した膜体が使用される。従って反応液がこの分
離膜（３）を通過する際に膜体の表面と内部の一方又は
双方に固定化された酵素Ｅの作用により第２段の反応が
生じ、最終生成物Ｃが生成される。またこの分離膜
（３）により反応液中の微生物又は酵素ｍ等は分離され
て不透過液側に濃縮され、再び反応槽（１）へ還流され
る。In FIG. 1, (1) is a reaction tank, (2) is a pump,
(3) is a separation membrane. There is a microorganism or enzyme m inside the reaction tank (1), and the stock solution A is converted into an intermediate product B by a first-stage reaction with the microorganism or enzyme m. The reaction liquid in which the intermediate product B, the stock solution A, and the microorganism or the enzyme m are mixed is sent to the separation membrane (3) by the pump (2). Enzyme E for causing one or both of the two to perform the second step reaction
A membrane body in which is immobilized is used. Therefore, when the reaction solution passes through the separation membrane (3), the second step reaction occurs due to the action of the enzyme E immobilized on one or both of the surface and the inside of the membrane, and the final product C is produced. It The microorganisms or enzymes m in the reaction solution are separated by the separation membrane (3), concentrated on the side of the impermeable liquid, and then recirculated to the reaction tank (1).
このようにして、本発明によれば二段反応を単一の反応
槽と分離膜のみを用いて行わせることができる。Thus, according to the present invention, the two-step reaction can be carried out using only a single reaction tank and a separation membrane.
なお酵素Ｅとしては目的とする反応により種々の酵素が
選択されるが、例えばエタノールから酢酸を製造する場
合には、アルコールデヒドロゲナーゼとアルデヒドデヒ
ドロゲナーゼを使用すればよい。このような反応用の酵
素を膜体に固定化させるには、膜体を化学物質で処理
後、酵素を共有結合によりその化学物質に結合させる化
学的吸着法や、膜体を酵素の水溶液に浸漬するか、膜体
に酵素の水溶液を圧入する物理的吸着法を取ることがで
きる。なお、膜体としては反応液中から微生物又は酵素
ｍ等を分離できる一般的な膜を使用することができ、例
えば市販のUF膜を使用すればよい。Various enzymes can be selected as the enzyme E depending on the intended reaction. For example, when acetic acid is produced from ethanol, alcohol dehydrogenase and aldehyde dehydrogenase may be used. To immobilize the enzyme for such a reaction on the membrane, a chemical adsorption method in which the membrane is treated with a chemical substance and then the enzyme is covalently bonded to the chemical substance, or the membrane is treated with an aqueous solution of the enzyme. It is possible to employ a physical adsorption method of dipping or press-fitting an aqueous solution of the enzyme into the membrane. As the membrane, a general membrane that can separate microorganisms or enzymes m from the reaction solution can be used, and for example, a commercially available UF membrane can be used.
（実施例） 次にグルコースをサッカロマイセス−セレビシエ（Sacc
haromyces-cerevisiae）協会７号菌を用いてエタノール
に変換し、更にアルコールデヒドロゲナーゼとアルデヒ
ドデヒドロゲナーゼを使用して酢酸を製造する二段反応
についての実施例を示す。(Example) Next, glucose was added to Saccharomyces cerevisiae (Sacc
An example of a two-step reaction in which acetic acid is produced by using alcohol dehydrogenase and aldehyde dehydrogenase after conversion into ethanol using Harmonyce-cerevisiae) No. 7 strain is shown.
「酵素固定化膜の製造方法」 まずセラミック膜（平均孔径0.2μ、膜面積50cm2）を60
0mlのトルエンに浸漬させ、沸騰石を添加後マントルヒ
ーターを用いて還流しながら加熱した。トルエンが沸騰
後、60mlのシラン剤（信越化学社製、γ−アミノプロピ
ルトリエトキシシラン）を添加し、４時間還流を行いな
がら加熱した。反応後この溶媒を捨て、トルエン臭がな
くなるまでセラミック膜をアセトンで洗浄した。"Method for producing enzyme-immobilized membrane" First, 60 ceramic membranes (average pore size 0.2μ, membrane area 50cm 2 )
It was immersed in 0 ml of toluene, and after adding boiling stone, it was heated under reflux using a mantle heater. After toluene was boiled, 60 ml of a silane agent (γ-aminopropyltriethoxysilane, manufactured by Shin-Etsu Chemical Co., Ltd.) was added, and the mixture was heated under reflux for 4 hours. After the reaction, this solvent was discarded, and the ceramic membrane was washed with acetone until the toluene odor disappeared.
このように処理したセラミック膜をエタノール中に浸漬
後、真空ポンプを用いて脱気を行ない、純水で洗浄し、
１％グルタルアルデヒド150mlを添加し、室温で２時間
静置した。その後、セラミック膜を無臭になるまで純水
で洗浄し、更に10％アルコールデヒドロゲナーゼと10％
アルデヒドデヒドロゲナーゼの混合液に24時間浸漬後、
純水で洗浄した。The ceramic membrane thus treated is immersed in ethanol, degassed using a vacuum pump, washed with pure water,
150 ml of 1% glutaraldehyde was added, and the mixture was left standing at room temperature for 2 hours. Then, the ceramic membrane is washed with pure water until it becomes odorless, and then 10% alcohol dehydrogenase and 10%
After soaking in a mixture of aldehyde dehydrogenase for 24 hours,
It was washed with pure water.
「醗酵及び反応」 実容量3lの醗酵槽にグルコース18％、酵母エキス２％と
なるように調整された培地を入れ、サッカロマイセス−
セレビシエ（Saccharomyces-cerevisiae）協会７号菌を
接種し、1 VVMの空気量で４〜６日間第１段の培養を行
った。エタノール濃度が９％（W/V）以上（収率95％以
上）に達した時点よりNADを最終濃度１％以上になるよ
うに添加した。その後醗酵槽から4l/minで培養液を引き
抜き、クロスフロー式で前記した分離膜に通して反応酵
素であるアルコールデヒドロゲナーゼとアルデヒドデヒ
ドロゲナーゼによりエタノールを酢酸に変換させる第２
段の反応を行わせると同時に濾過させ、不透過液（濃縮
液）は醗酵槽に戻す連続濾過培養を滞留時間96時間で行
った。"Fermentation and reaction" Put a medium adjusted to 18% glucose and 2% yeast extract into a fermentor with a real volume of 3 liters, and add Saccharomyces-
C. cerevisiae (Saccharomyces-cerevisiae) Society No. 7 was inoculated and the first stage culture was carried out at an air volume of 1 VVM for 4 to 6 days. When the ethanol concentration reached 9% (W / V) or more (yield 95% or more), NAD was added so that the final concentration was 1% or more. After that, the culture solution is drawn out from the fermenter at 4 l / min and passed through the separation membrane in a cross-flow system to convert ethanol to acetic acid by the reaction enzymes alcohol dehydrogenase and aldehyde dehydrogenase.
At the same time as carrying out the stage reaction, filtration was carried out, and the impermeable liquid (concentrated liquid) was returned to the fermentor for continuous filtration culture with a residence time of 96 hours.
その結果、濾過中の生成酢酸量は培養液中のエタノール
の95％以上が変換された量となり、未反応のエタノール
濃度は培養液中のエタノール濃度の５％以下になった。As a result, the amount of acetic acid produced during the filtration was an amount obtained by converting 95% or more of ethanol in the culture medium, and the unreacted ethanol concentration was 5% or less of the ethanol concentration in the culture medium.
（発明の効果） 本発明は以上に説明したように、第１段の反応を行わせ
た反応液を表面及び／又は内部に酵素を固定化した膜体
に送って第２段の反応と微生物又は酵素の分離とを同時
に行わせるようにしたもので、これにより単一の反応槽
及び分離膜を用いて二段反応をおこなわせることができ
る。なお、本発明は実施例の酢酸製造プロセスに限定さ
れるものではなく、その他の二段反応プロセスに広く応
用できることはいうまでもない。よって本発明は従来の
問題点を解決した二段反応法として、産業の発展に寄与
するところは極めて大きいものがある。(Effects of the Invention) As described above, the present invention sends the reaction solution in which the reaction of the first step has been carried out to the membrane having the enzyme immobilized on the surface and / or the inside thereof, and the reaction of the second step and the microorganism. Alternatively, the enzyme is separated at the same time, which allows a two-step reaction to be carried out using a single reaction tank and a separation membrane. Needless to say, the present invention is not limited to the acetic acid production process of the examples, and can be widely applied to other two-step reaction processes. Therefore, the present invention, as a two-step reaction method that solves the conventional problems, has an extremely great contribution to the industrial development.
第１図は本発明の二段反応法を説明するフローシート、
第２図は従来の二段反応法を説明するフローシートであ
る。 （１）：反応槽、（３）：分離膜FIG. 1 is a flow sheet for explaining the two-step reaction method of the present invention,
FIG. 2 is a flow sheet for explaining the conventional two-step reaction method. (1): Reaction tank, (3): Separation membrane
Claims (1)
を行わせた反応液を表面と内部の一方又は双方に酵素を
固定化した膜体に送り、この膜体の酵素の作用により第
２段の反応を行わせると同時に膜体により反応液中の微
生物又は酵素を分離し、微生物又は酵素を含む不透過液
を第１段の反応槽へ還流させるとともに最終生成物を外
部に取り出すことを特徴とする二段反応法。1. A reaction solution in which a first-stage reaction is carried out by the action of a microorganism or an enzyme is sent to a membrane on which an enzyme is immobilized on one or both of the surface and the inside, and by the action of the enzyme on the membrane, At the same time as carrying out the two-stage reaction, the microorganisms or enzymes in the reaction liquid are separated by the membrane, and the impermeable liquid containing the microorganisms or enzymes is refluxed to the first-stage reaction tank and the final product is taken out to the outside. A two-step reaction method characterized by.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11247789A JPH0793877B2 (en) | 1989-05-01 | 1989-05-01 | Two-step reaction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11247789A JPH0793877B2 (en) | 1989-05-01 | 1989-05-01 | Two-step reaction method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02291282A JPH02291282A (en) | 1990-12-03 |
| JPH0793877B2 true JPH0793877B2 (en) | 1995-10-11 |
Family
ID=14587616
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11247789A Expired - Lifetime JPH0793877B2 (en) | 1989-05-01 | 1989-05-01 | Two-step reaction method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0793877B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2777803B1 (en) * | 1998-04-24 | 2000-07-28 | Univ Claude Bernard Lyon | PROCESS FOR THE ACTIVE AND SELECTIVE REMOVAL OF SMALL MOLECULES BY ENZYMATIC PUMPING: ACTIVE DIALYSIS |
-
1989
- 1989-05-01 JP JP11247789A patent/JPH0793877B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02291282A (en) | 1990-12-03 |
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