JPH0242992A - Reaction using biocatalyst and device for said reaction - Google Patents

Reaction using biocatalyst and device for said reaction

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Publication number
JPH0242992A
JPH0242992A JP19401088A JP19401088A JPH0242992A JP H0242992 A JPH0242992 A JP H0242992A JP 19401088 A JP19401088 A JP 19401088A JP 19401088 A JP19401088 A JP 19401088A JP H0242992 A JPH0242992 A JP H0242992A
Authority
JP
Japan
Prior art keywords
reaction
biocatalyst
product
tank
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
Application number
JP19401088A
Other languages
Japanese (ja)
Other versions
JP2520155B2 (en
Inventor
Tetsuya Tosa
土佐 哲也
Masaru Senuma
瀬沼 勝
Nobuyuki Sakata
信行 坂田
Noriyuki Nishimura
西村 紀之
Masakatsu Furui
古井 正勝
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tanabe Seiyaku Co Ltd
Original Assignee
Tanabe Seiyaku Co Ltd
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Filing date
Publication date
Application filed by Tanabe Seiyaku Co Ltd filed Critical Tanabe Seiyaku Co Ltd
Priority to JP63194010A priority Critical patent/JP2520155B2/en
Publication of JPH0242992A publication Critical patent/JPH0242992A/en
Application granted granted Critical
Publication of JP2520155B2 publication Critical patent/JP2520155B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

PURPOSE:To make possible to directly use biocatalyst without depression of activity accompanying immobilization by separating reacted solution of substrate and biocatalyst to the biocatalyst and a liquid containing substrate and reacted product by ultrafiltration membrane, the returning the former to reaction tank, etc. CONSTITUTION:Substrate is reacted with biocatalyst in aqueous solution in reaction tank 1 and the reacted solution is sent to ultrafilter 2. The reacted solution is separated to the biocatalyst and a solution containing reacted product and residual substrate by ultrafiltration membrane in the ultrafilter, then the former is returned to the reaction tank 1 and the latter is sent to crystallizing tank 3. The reacted product is crystallized in the crystallizing tank 3 and a solution containing the crystallized substance and the substrate is sent to a filter 4. Then, filtered solution containing the substrate is returned to the reaction tank 1 and crystallized reacted product is accumulated in the filter 4. Said process is preferably performed for transaminase as biocatalyst, phenylpyruvic acid and L-asparagine acid as substrate and L-phenylalanine as reacted product.

Description

【発明の詳細な説明】 [産業上の利用分野] 本発明は、酵素や微生物菌体のような生体触媒を基質と
反応させて種々の有用物質を製造する方法において、該
触媒反応を効率良く行う方法および該方法に用いるため
の装置に関するものである。
Detailed Description of the Invention [Industrial Application Field] The present invention is a method for producing various useful substances by reacting a biocatalyst such as an enzyme or a microbial cell with a substrate, in which the catalytic reaction is efficiently carried out. The present invention relates to a method for performing the method and an apparatus for use in the method.

さらに詳しくは、本発明は、限外濾過膜を併置した反応
部と、生成物の分離機能を備えた晶析部とで構成される
反応装置を用い、反応混合物を該装置に循環させて生成
物を析出させ、分離することにより、高い基質濃度の反
応混合物から生成物を蓄積させ、効率よく生体触媒反応
を行う方法およびそのための装置に関するものである。
More specifically, the present invention uses a reaction device consisting of a reaction section in which an ultrafiltration membrane is juxtaposed and a crystallization section equipped with a product separation function, and circulates the reaction mixture through the device to generate the product. The present invention relates to a method for efficiently carrying out a biocatalytic reaction by accumulating products from a reaction mixture with a high substrate concentration by precipitating and separating the substances, and an apparatus therefor.

[従来技術5と解決すべき課題] 酵素や微生物を触媒とする反応は、その高い基質特異性
により、常温、常圧の緩和な条件下で効率良(進むこと
から、これらの生体触媒を用いて種々の有用物質を製造
する試みがなされている。
[Prior art 5 and issues to be solved] Reactions using enzymes or microorganisms as catalysts proceed efficiently under mild conditions at room temperature and pressure due to their high substrate specificity; therefore, it is difficult to use these biocatalysts. Attempts have been made to produce various useful substances.

これらの生体触媒を用いる反応は通常、水溶液または水
性懸濁液中で行なわれるが、これら生体触媒は、高温等
の過酷な条件下では極めて容易に失活してしまうので、
反応後、生成物を懸濁状の反応混合物から分離し、さら
に生体触媒をその活性を維持したままで回収して再刊用
することは極めて困難である。従って、たとえ充分な活
性か残っていても反応液と一緒に捨てなければならず、
極めて不経済であった。
Reactions using these biocatalysts are usually carried out in aqueous solutions or suspensions, but these biocatalysts are extremely easily deactivated under harsh conditions such as high temperatures.
After the reaction, it is extremely difficult to separate the product from the suspended reaction mixture and recover the biocatalyst while maintaining its activity for reprinting. Therefore, even if there is sufficient activity remaining, it must be discarded together with the reaction solution.
It was extremely uneconomical.

近年、上記問題の解決策として、固定化生体触媒の利用
が高まっている。固定化生体触媒とは、生体触媒を、適
当な固体担体に眼前させるか、もしくは固体担体に包括
せしめてなる不溶性の触媒である。この固定化生体触媒
を用いた物質生産プロセスの利点として、生体触媒が安
定化されており、反応の連続操作が可能であること、生
成物と生体触媒との分離が容易であり、澄明な反応液が
得られること、生体触媒の反IV刊用か可能であること
等を挙げ得るか、他方、欠点として、固定化時に生体触
媒の一部変性および失活を免れ得す活性か低下している
こと、また、このような生体触媒反応の反応性は基質濃
度に依存しているが、基質濃度を高くすると生成物が飽
和溶解度以上に生成し、触媒内部や表面に析出して反応
を円滑に行えなくなるという点か指摘される。即ち、多
くの固定化生体触媒反応では、蓄積できる生成物の濃度
は、それ自身の反応温度における飽和溶解度によって決
まるために、一般には飽和溶解度以上の生成物を得るこ
とができなかったのである。
In recent years, the use of immobilized biocatalysts has been increasing as a solution to the above problems. An immobilized biocatalyst is an insoluble catalyst in which the biocatalyst is placed in front of a suitable solid carrier or is enclosed in a solid carrier. The advantages of this substance production process using an immobilized biocatalyst are that the biocatalyst is stabilized, the reaction can be operated continuously, the product and the biocatalyst can be easily separated, and the reaction is clear. On the other hand, the drawbacks are that the biocatalyst can be partially denatured and deactivated during immobilization, resulting in decreased activity. In addition, the reactivity of such biocatalytic reactions depends on the substrate concentration, but when the substrate concentration is increased, the product is generated above the saturation solubility and precipitates inside or on the catalyst surface, making the reaction smoother. It has been pointed out that this means that they will no longer be able to do so. That is, in many immobilized biocatalyst reactions, the concentration of the product that can be accumulated is determined by the saturation solubility at its own reaction temperature, so it has generally been impossible to obtain a product with a saturation solubility or higher.

このような状況下、固定化生体触媒反応での生産効率を
向上する手段として、基質や生成物等の低分子重物質は
透過させるか、生体触媒等の高分子量物質を透過させな
いような膜を用いて生体触媒を隔離することにより、全
体として固定化生体触媒作用を発揮させ、生体触媒の固
定化操作に伴う活性の低下を防ぐ方法か提案されている
。しかしながら、この方法では、活性は充分に発現され
得るか、安定性は従来の吸着法や包括法で調製された固
定化生体触媒の安定性に比べて低く、失活し易いために
、回収再利用には不適当である。他方、本願出願人は、
固定化生体触媒を用いる反応において、反応の進行と同
時に生成物の分離、精製を行うことにより、反応液中の
生成物濃度を高める方法を開示した(特開昭61−57
89号)。
Under these circumstances, as a means to improve the production efficiency of immobilized biocatalyst reactions, we have developed membranes that either allow low-molecular weight substances such as substrates and products to pass through, or that do not allow high-molecular weight substances such as biocatalysts to pass through. It has been proposed that by isolating the biocatalyst using a biocatalyst, the effect of the immobilized biocatalyst can be exerted as a whole, thereby preventing a decrease in activity due to the immobilization operation of the biocatalyst. However, with this method, the activity cannot be sufficiently expressed, and the stability is lower than that of immobilized biocatalysts prepared by conventional adsorption methods or entrapment methods, and it is easy to deactivate. It is unsuitable for use. On the other hand, the applicant is
In a reaction using an immobilized biocatalyst, a method was disclosed for increasing the concentration of the product in the reaction solution by separating and purifying the product simultaneously with the progress of the reaction (Japanese Unexamined Patent Publication No. 61-57
No. 89).

この方法によって、生成物の高濃度化は達成されたが、
固定化に伴う活性の低下という問題点は未解決のままで
あった。従って、本発明者らは、より効率のよい生体触
媒による反応方法を開発することを目的として鋭意、研
究を謙「ねできた。
Although a high concentration of the product was achieved by this method,
The problem of decreased activity due to immobilization remained unsolved. Therefore, the present inventors have been able to carry out extensive research with the aim of developing a more efficient reaction method using a biocatalyst.

[課題を解決するための手段] 本発明は、上記の様々な課題を解決するものであって、
生体触媒の活性を高く維持しつつ、固定化生体触媒の機
能を充分に発揮させ、高濃度の生成物を蓄積させて効率
よく反応させる方法および該方法に用いるための装置を
提供するものである。
[Means for solving the problems] The present invention solves the various problems mentioned above,
The present invention provides a method for efficiently reacting by accumulating highly concentrated products by fully demonstrating the function of an immobilized biocatalyst while maintaining a high activity of the biocatalyst, and an apparatus for use in the method. .

本発明方法は、生体触媒を透過させない限外濾過膜(以
下、UF膜と弥する)を併置する反応部と、生成物の析
出、分離機能を備えた晶析部とからなる反応装置を用い
ることによって達成された。
The method of the present invention uses a reaction apparatus consisting of a reaction section in which an ultrafiltration membrane (hereinafter referred to as UF membrane) that does not allow biocatalysts to pass is placed in parallel, and a crystallization section equipped with product precipitation and separation functions. This was achieved by

即ち、本発明は、基質と生体触媒とを反応させて有用物
質を製造する方法において、反応混合物をUF膜に通し
て生成物に富む濾液を分離し、該濾液から生成物を析出
、蓄積させることを特徴とする方法、並びに該方法に用
いるための装置を提供するものである。
That is, the present invention provides a method for producing useful substances by reacting a substrate and a biocatalyst, in which a reaction mixture is passed through a UF membrane to separate a product-rich filtrate, and the product is precipitated and accumulated from the filtrate. The present invention provides a method characterized by the above, and an apparatus for use in the method.

本発明方法は、たとえば、撹拌下、反応部の反応槽内で
基質と生体触媒とを反応させながら、これら出発物質と
生成物とを含有する反応混合物を循環させてUF膜に接
触させ、生成物に富む濾液を分離し、該濾液を、生成物
の溶解度が反応部での飽和溶解度よりも低くなるように
温度設定された晶析槽に送って析出させ、得られた生成
物を分離し、蓄積させた後、母液を再ひ反応部に戻して
再使用することで実施される。
In the method of the present invention, for example, while a substrate and a biocatalyst are reacted in a reaction tank of a reaction part under stirring, a reaction mixture containing these starting materials and products is circulated and brought into contact with a UF membrane, thereby producing a product. The product-rich filtrate is separated, the filtrate is sent to a crystallization tank whose temperature is set so that the solubility of the product is lower than the saturated solubility in the reaction zone, and the resulting product is separated. After accumulation, the mother liquor is returned to the reaction section for reuse.

本発明装置の反応槽に併置されるOF膜としては、生体
触媒である微生物細胞や酵素タンパク質の透過を妨げ、
低分子量の基質や生成物のみを透過させる適当な孔径の
細孔膜である限り、その種類に制限なく、平膜、中空糸
(ホローファイバー)膜等から任意に選択することかで
きる。しかしながら、分画分子型か約5.000〜50
,000であって、単位体積当たりの透過面積か約2な
いし30m2と大きいホローファイバー型式の膜が好ま
しい。
The OF membrane placed in parallel with the reaction tank of the device of the present invention is designed to prevent the permeation of microbial cells and enzyme proteins that are biocatalysts.
As long as the membrane has a suitable pore size that allows only low-molecular-weight substrates and products to pass through, the membrane is not limited in type and can be arbitrarily selected from flat membranes, hollow fiber membranes, and the like. However, the fractionated molecular type is about 5,000 to 50
,000 and a large hollow fiber type membrane with a permeation area of about 2 to 30 m2 per unit volume is preferred.

反応槽としては、基質と生体触媒間の外部拡散抵抗を除
去するための機能を備えたものであればいずれの型式の
ものであってもよく、適当な撹拌機の付いた撹拌槽や、
液流動用ポンプの付いた基型式のものを用いることかで
きる。
The reaction tank may be of any type as long as it has a function to remove external diffusion resistance between the substrate and the biocatalyst, such as a stirring tank with an appropriate stirrer,
A basic model with a pump for liquid flow can be used.

また、晶析槽としては、熱交換機能、固−液分離機能、
撹拌機能を有するものであれば、いかなる型式のものを
選択してもよい。
In addition, the crystallization tank has heat exchange function, solid-liquid separation function,
Any type may be selected as long as it has a stirring function.

上記の如く、本発明方法では、反応槽〜UF膜、および
UF膜〜晶析槽〜反応槽間に、反応液を循環させる。従
って、本発明装置は、これらの各部位を送液ラインで連
結すると共に、液を循環させ°るための循環ポンプを適
宜配設して構成されでい本発明方法は、通常の生体触媒
による反応に広く適用可能である。そのような反応には
、たとえば、トランスアミナーゼのような酵素を粗酵素
のまま、または精製酵素として用いることができ、ある
いは、トランスアミナーセ活性を有するパラコツカス0
デニトリフイカンス(P aracoccusdeni
trificans) I F O12442やエッ/
エリヒア・コリ(E 5cherichia cal 
i) P A 5 Q 1株(特願昭62−31350
5号)の如き微生物細胞の培養液をそのまま、あるいは
濃縮精製した濃縮菌体として用いることもできる。この
ような生体触媒は市販されているか、または調製可能で
あるか、微生物細胞を用いるときには、反応液中に培養
液由来の夾雑物か混入することを避けると共に、取り扱
いを容易にするために、培養液を115〜1750程度
に濃縮精製したものを用いることが好ましい。
As described above, in the method of the present invention, the reaction liquid is circulated between the reaction tank and the UF membrane, and between the UF membrane, the crystallization tank, and the reaction tank. Therefore, the device of the present invention cannot be constructed by connecting these parts with a liquid supply line and appropriately disposing a circulation pump for circulating the liquid. It is widely applicable to reactions. For such reactions, for example, enzymes such as transaminases can be used in their crude form or as purified enzymes, or
Denitrificans (Paracoccus deni)
trificans) I F O12442 and E/
E5cherichia cal
i) P A 5 Q 1 share (Patent application 1986-31350
A culture solution of microbial cells such as No. 5) can be used as it is, or as concentrated and purified microbial cells. Is such a biocatalyst commercially available or can it be prepared?When using microbial cells, in order to avoid contamination of the reaction solution with contaminants derived from the culture solution and to facilitate handling, It is preferable to use a culture solution that has been concentrated and purified to a concentration of about 115 to 1750.

また、基質は、目的物質に対応するものが選択されるが
、それが水溶性の場合には、適当に高濃度の水溶液とし
て用い、難溶性であれば、懸濁液として用いる。反応槽
への添加は、最初に一括して加えるか、あるいは反応中
に数回に分けて加えるいずれの方法でもよい。生体触媒
反応が基質で阻害される場合には後者の方法が好ましい
The substrate is selected to correspond to the target substance; if the substance is water-soluble, it is used as an appropriately concentrated aqueous solution, and if it is poorly soluble, it is used as a suspension. The material may be added to the reaction tank either all at once at the beginning or in several portions during the reaction. The latter method is preferred when the biocatalytic reaction is inhibited by the substrate.

本発明方法では、上記の如く、反応槽内の反応混合物を
重環させてUF膜に接触させ、生成物に富む濾液と生体
触媒を含有する反応液とを分離する。後者はそのまま反
応槽に戻されるが、前者の濾液を晶析槽に導き、該槽内
で生成物を析出させて分離した後、反応母液を反応槽に
戻す。この−連の反応機構を通して循環される反応液の
流速は、反応槽〜UF膜間では生体触媒がUF膜内に貯
溜されない程度であればよ(、UF膜〜晶析槽〜反応槽
間では、反応効率の観点から、UF膜の濾液中に基質が
若干残存している状態が得られる程度の流速とするのか
好ましい。
In the method of the present invention, as described above, the reaction mixture in the reaction tank is subjected to polycyclization and brought into contact with the UF membrane to separate the product-rich filtrate from the reaction liquid containing the biocatalyst. The latter is returned to the reaction tank as it is, but the filtrate of the former is led to a crystallization tank, in which the product is precipitated and separated, and then the reaction mother liquor is returned to the reaction tank. The flow rate of the reaction solution circulated through this series of reaction mechanisms is only required to be such that the biocatalyst is not stored in the UF membrane between the reaction tank and the UF membrane (and between the UF membrane, crystallization tank and the reaction tank). From the viewpoint of reaction efficiency, it is preferable to set the flow rate to such an extent that a state in which a small amount of the substrate remains in the filtrate of the UF membrane is obtained.

反応槽の温度は生体触媒の活性と安定性を維持するため
に、通常、0〜60°Cの範囲とするが、20〜45°
Cに設定することが好ましい。
In order to maintain the activity and stability of the biocatalyst, the temperature of the reaction tank is usually in the range of 0 to 60°C, but it is 20 to 45°C.
It is preferable to set it to C.

晶析槽の温度は、反応温度における生成物の飽和溶解度
よりも低い飽和溶解度を与えるt温度に設定することか
必要である。即ち、溶解度が温度上昇に伴って上昇する
物質の場合には、晶析槽の温度を反応槽の温度よりも低
くし、その逆の場合には高くすればよい。
It is necessary that the temperature of the crystallizer be set at a temperature that gives a saturation solubility lower than the saturation solubility of the product at the reaction temperature. That is, in the case of a substance whose solubility increases with increasing temperature, the temperature of the crystallization tank may be lower than the temperature of the reaction tank, and vice versa, it may be set higher.

次に、上記本発明方法の1実施態様を、図面に従って説
明する。
Next, one embodiment of the method of the present invention will be described with reference to the drawings.

第1図は本発明の反応装置の該略ブロック図である。反
応槽(1)にはホローファイバー型のOF膜(2)か、
晶析槽(3)には濾過器(4)がそれぞれ併設されてお
り、これらの間にはポンプ(5)、(6)および(7)
が配設され、反応槽(1)からUF膜(2)には反応K
1合物力、UF膜(2)カラ晶析槽(3)ニハUFII
!a濾液が、そして濾過器(4)から反応槽(1)間に
は生成物分離後の母液が夫々送液ライン(10)、(t
o’)、(10”)を通って循環するように構成されて
いる。
FIG. 1 is a schematic block diagram of the reactor of the present invention. The reaction tank (1) is equipped with a hollow fiber type OF membrane (2) or
A filter (4) is attached to each crystallization tank (3), and pumps (5), (6), and (7) are installed between them.
is arranged, and reaction K is supplied from the reaction tank (1) to the UF membrane (2).
1 Combined strength, UF membrane (2) Color crystallization tank (3) Niha UFII
! The filtrate a is transferred between the filter (4) and the reaction tank (1), and the mother liquor after product separation is transferred through the liquid feeding lines (10) and (t), respectively.
o'), (10'').

反応槽(1)および晶析槽(3)にはそれぞれ、撹拌機
(8)および(9)か設けられている。晶析槽(3)に
併置される濾過器(4)の形状および設置方法は特に限
定されるものでなく、第1図に示すように、晶析槽(4
)にオーバーフロー管を付け、その管の出口に設置して
もよいが、晶析槽(3)の断面全域に吸引式の平板式濾
過板を設置したり、あるいは、円筒式濾過板を晶析槽(
3)内に浸漬して設ける方法(第2図参照)てもよい。
The reaction tank (1) and the crystallization tank (3) are provided with stirrers (8) and (9), respectively. The shape and installation method of the filter (4) placed in parallel with the crystallization tank (3) are not particularly limited, and as shown in FIG.
) may be attached to the overflow pipe and installed at the outlet of the pipe, but it is also possible to install a suction type flat filter plate over the entire cross section of the crystallization tank (3), or to install a cylindrical filter plate at the outlet of the pipe. Tank (
3) may be provided by immersing it in the interior (see Fig. 2).

本発明装置を用いて生体触媒反応を行うには、まず、反
応槽(1)に基質溶液または基質懸濁液と生体触媒を入
れ、反応混合物を撹拌機(8)で撹拌する。
To perform a biocatalytic reaction using the apparatus of the present invention, first, a substrate solution or substrate suspension and a biocatalyst are placed in a reaction tank (1), and the reaction mixture is stirred with a stirrer (8).

同時に、反応混合物を、ポンプ(5)により送液ライン
(lO)を経てOF膜(2)に導く。UF膜(2)がホ
ローファイバー膜であれば、循環液を膜の内、外いずれ
に導いてもよい。次いで、UF膜(2)を透過した濾液
をポンプ(6)によって抜き出し、送液ライン(10’
)を経て晶析槽(3)に送り込むと、反応生成物の溶解
度が反応槽(1)内におけるよりも低くなるように温度
設定された晶析槽(3)内で生成物は過飽和となり、析
出する。生成物が析出し、反応槽(1)内よりも低濃度
で飽和されたUF膜(2)濾液はオーバーフローして濾
過機(4)に入り、生成物が分離される。析出した生成
物を晶析部に残し、母液をポンプ(7)により送液ライ
ン(10′”)を経て反応14!(1)に戻すと、該液
は未飽和の基質を溶解し、再度反応に供せられる。
At the same time, the reaction mixture is guided by the pump (5) through the liquid delivery line (IO) to the OF membrane (2). If the UF membrane (2) is a hollow fiber membrane, the circulating fluid may be guided either inside or outside the membrane. Next, the filtrate that has passed through the UF membrane (2) is extracted by the pump (6), and the filtrate is transferred to the liquid feeding line (10').
) and into the crystallization tank (3), the product becomes supersaturated in the crystallization tank (3) whose temperature is set so that the solubility of the reaction product is lower than that in the reaction tank (1). Precipitate. The product is precipitated, and the filtrate of the UF membrane (2), which is saturated at a lower concentration than in the reaction tank (1), overflows and enters the filter (4), where the product is separated. The precipitated product is left in the crystallization section, and the mother liquor is returned to the reaction 14! (1) by the pump (7) via the liquid supply line (10'''), whereupon the unsaturated substrate is dissolved and the mother liquor is again Subjected to reaction.

基質が消耗されるまでこの操作を繰り返して行うと、晶
析部には懸濁状の生成物のみが蓄積されることになる。
If this operation is repeated until the substrate is consumed, only suspended product will accumulate in the crystallization section.

第2図は他の実施例を示す図であって、第1図の反応装
置において、反応槽(1)か除かれている。
FIG. 2 is a diagram showing another embodiment, in which the reaction vessel (1) is removed from the reaction apparatus of FIG. 1.

この場合には、基質を晶析槽(3)内に入れ、生体触媒
をOF膜(2)の空間に置いて、反応混合物を晶析槽(
3)からUF膜(2)に循環させて反応させる。
In this case, the substrate is placed in the crystallizer (3), the biocatalyst is placed in the space of the OF membrane (2), and the reaction mixture is placed in the crystallizer (3).
3) to the UF membrane (2) for reaction.

この装置では、晶析部[晶析槽(3)と濾過器(4)]
の実効容積に対する反応部[UF膜(2)]の実効容積
の比か小さいので、反応液への生成物量の損失を少なく
することかできる。
In this device, the crystallization section [crystallization tank (3) and filter (4)]
Since the ratio of the effective volume of the reaction section [UF membrane (2)] to the effective volume of the UF membrane (2) is small, the loss of the amount of product to the reaction liquid can be reduced.

[実施例] 以下に実施例および実験例を挙げ、本発明をさらに詳し
く説明する。
[Example] The present invention will be explained in more detail by referring to Examples and Experimental Examples below.

実施例1 トランスアミナーセ活性を有するDNA組換
えエッシエリヒア・コリPΔ501株によるフェニルピ
ルビン酸とL−アスパラキン酸カらのし一フェニルアラ
ニンの生産 (1)装置 ホローファイバー型UF膜(旭化成、5IPI013、
透過面積02m2、万両分子ff16000)を併置し
た反応槽(実効容積0.55のと実効容積0.4512
の外浴付き晶析槽および円筒型平板濾過器(直径8cm
φガラスフィルター)を第1図の様式で連結したちのを
用いる。
Example 1 Production of phenylpyruvic acid and L-aspartic acid and phenylalanine by DNA recombinant Escherichia coli PΔ501 strain having transaminase activity (1) Apparatus Hollow fiber type UF membrane (Asahi Kasei, 5IPI013,
A reaction tank (with an effective volume of 0.55 and an effective volume of 0.4512
Crystallization tank with external bath and cylindrical flat plate filter (diameter 8cm)
φ glass filters) are connected in the manner shown in Figure 1.

(2)生体触媒 微生物細胞を次の如く調製した。(2) Biocatalyst Microbial cells were prepared as follows.

まず、グルコース0.2%、ラクトース1%、コーンス
チーブリカ−2%、ミーストN2%、クルタミン酸ナト
リウム0.5%、(NH,)、30.01%、K、HP
O,0,7%、KH,PO,0,3%、Mg S O4
・7HtO0,025%、カラリン0.03%を含む培
地I Q12(pH7,0)にDNA組換えエノシェリ
ヒア・コリPA501株(宿主:エッンエリヒア・コリ
HB101株;ベクター・pUC18;挿入DNA・バ
ラコツカス・デニトリフィカンスのゲノムDNAの一部
)の前項er(10,1ρを植菌し、37°Cて22h
通気撹拌培養した。
First, glucose 0.2%, lactose 1%, corn stew liqueur 2%, meat N 2%, sodium curtamate 0.5%, (NH,), 30.01%, K, HP
O, 0.7%, KH, PO, 0.3%, Mg SO4
・DNA recombinant E. coli strain PA501 (host: E. coli HB101 strain; vector, pUC18; inserted DNA, Baracoccus denitori Part of the genomic DNA of C. ficans) was inoculated with er (10,1ρ) and kept at 37°C for 22 hours.
Culture was carried out with aeration and stirring.

培養液を遠心分離してl/37に濃縮し、濃縮微生物細
胞を調製した。
The culture solution was centrifuged and concentrated to l/37 to prepare concentrated microbial cells.

(3)操作 濃W6 ?a 生物細胞59、フェニルピルビン酸ナト
Jウム(1水塩)0.2moL L−アスパラギン酸ア
ンモニウムI−625moQを反応槽に充填し全量を0
、97Q(pr(8,0)とした。
(3) Operation depth W6? a Biological cells 59, sodium phenylpyruvate J (monohydrate) 0.2 moL ammonium L-aspartate I-625 moQ were charged into the reaction tank and the total amount was reduced to 0.
, 97Q(pr(8,0)).

反応槽の温度を30’Cに設定し、撹拌を行うと同時に
?’?内の基質と微生物細胞をUF膜の外側(シェル側
)を循環させた。
Set the temperature of the reaction tank to 30'C and stir at the same time? '? The substrate and microbial cells inside were circulated around the outside (shell side) of the UF membrane.

同時に、UF膜から濾液を0.!M/hの流速で抜き出
し、5°Cに設定した晶析槽に送り、晶析槽からのオー
バフロー液は濾過器を通して反応槽に戻した。
At the same time, remove the filtrate from the UF membrane at 0. ! It was extracted at a flow rate of M/h and sent to a crystallization tank set at 5°C, and the overflow liquid from the crystallization tank was returned to the reaction tank through a filter.

反応開始0.5時間後に反応槽に0.02mo(のフェ
ニルピルビン酸ナトリウムを加え、以後05時間間隔で
同量のフェニルピルビン酸ナトリウムを加えて行き、7
5時間で総ff10.5mof!を仕込んだ。
0.5 hours after the start of the reaction, 0.02 mo (of sodium phenylpyruvate) was added to the reaction tank, and thereafter the same amount of sodium phenylpyruvate was added at intervals of 0.5 hours.
Total ff10.5mof in 5 hours! I prepared it.

24時間で反応を停止したところ、反応液中のフェニル
ピルビン酸は完全に消失し、0.486mof2のし一
フェニルアラニンが生成していた。
When the reaction was stopped after 24 hours, phenylpyruvic acid in the reaction solution completely disappeared, and 0.486 mof2 of phenylalanine was produced.

生成したし一フェニルアラニンのうち57%は固を目に
、残りの43%か液相に存在していた。
Of the produced di-phenylalanine, 57% was present in the solid phase, and the remaining 43% was present in the liquid phase.

この固相のL−フェニルアラニンを瘍刑し、その時得ら
れた母液と洗滌液(0,98512)に新たに濃縮微生
物細胞5gとフェニルピルビン酸ナトリウムを加え(初
回0.05mo(、以後は0.5時間毎に0.02mo
f!、総仕込ff10.25moの、pHを80として
24時間反応させ、1回目の反応と同様に、蓄積したし
一フェニルアラニンの結晶を濾別した。この操作を合計
3回行った(フェニルピルビン酸の総仕込fft1.2
5moのところ、生成したI、−フェニルアラニンは1
.185moQ(転換率911.5%)となり、このう
ち76.5%が結晶として回収できた。
This solid phase L-phenylalanine was crushed, and 5 g of concentrated microbial cells and sodium phenylpyruvate were added to the mother liquor and washing solution (0.98512) obtained at that time (0.05 mo for the first time (0.05 mo for the first time, 0.05 mo for the first time). 0.02 mo every 5 hours
f! A total of ff10.25 mo was reacted for 24 hours at a pH of 80, and the accumulated monophenylalanine crystals were filtered out in the same manner as the first reaction. This operation was performed a total of 3 times (total amount of phenylpyruvic acid fft1.2
5mo, the produced I,-phenylalanine is 1
.. The result was 185 moQ (conversion rate 911.5%), of which 76.5% was recovered as crystals.

実施例2 トランスアミナーゼ活性を有するパラコツカ
ス・デニトリフィカンスIF○124,42によるフェ
ニルピルビン酸とL−アスパラキン酸からのし一フェニ
ルアラニンの生産 (1)装置 ホローファイバー型UF膜(アミコン社、Hlpto−
s、透過面積0.083m’、分画分子↑10、 OO
O)と実効容積0.1712の外温付晶析槽とを第2図
の様式に従って連結して用いる。
Example 2 Production of phenylalanine from phenylpyruvic acid and L-aspartic acid by Paracoccus denitrificans IF○124,42 having transaminase activity (1) Apparatus Hollow fiber type UF membrane (Amicon, Hlpto-
s, transmission area 0.083 m', fractionated molecules ↑10, OO
O) and an externally heated crystallization tank with an effective volume of 0.1712 are connected and used according to the format shown in FIG.

(2)生体触媒 微生物細胞を次の如(調製した。(2) Biocatalyst Microbial cells were prepared as follows.

まず、グルコース1%、(NH4)28 P 040.
2%、ミーストN(ビール酵母エキス)1%、コーンス
チーブリ力−1%、ペプトン0.5%、K H2PO,
0,]%、Mg5O,・7820005%、カラリン0
103%を含む培地5ρ(pH7,0)にパラコツカス
・デニトリフィカンス+rO12442の前培養液0.
0 !Mを植菌し、30’Cて24時間通気撹拌培養し
た。培養液に1%のセチルトリメチルアンモニウムブロ
マイドをO,O!M添加し、30°Cで0.5時間放置
して微生物細胞の活性化を行った後、遠心分離によって
1/15に濃縮し、濃縮微生物細胞を調製した。
First, glucose 1%, (NH4)28P 040.
2%, Meest N (brewer's yeast extract) 1%, Cornsteeply-1%, peptone 0.5%, K H2PO,
0, ]%, Mg5O, 7820005%, Kararin 0
A preculture of Paracoccus denitrificans + rO12442 in a medium 5ρ (pH 7,0) containing 103% 0.
0! M was inoculated and cultured with aeration and stirring at 30'C for 24 hours. Add 1% cetyltrimethylammonium bromide to the culture solution O, O! After activating the microbial cells by adding M and leaving them at 30°C for 0.5 hours, the cells were concentrated to 1/15 by centrifugation to prepare concentrated microbial cells.

(3)操作 この濃縮微生物細胞169に水を加えて全量を0.03
1dとし、30°Cに保温したOF膜の外側空間部に充
填した。他方、晶析槽には、0.064 mo(2のフ
ェニルピルビン酸すトリウム(l水塩)、0.204m
o(!のL−アスパラキン酸アンモニウム、2 ×10
−5mo(のピリドキサル5゛−リン酸(補酵素)を充
填し、水で全量を0.16!Mとした。
(3) Operation Add water to this concentrated microbial cell 169 to bring the total volume to 0.03
1d, and filled into the outer space of the OF membrane kept at 30°C. On the other hand, in the crystallization tank, 0.064 mo (2 sodium phenylpyruvate (l hydrate), 0.204 m
o (! of L-ammonium aspartate, 2 × 10
-5mo(pyridoxal 5'-phosphate (coenzyme)) was charged, and the total amount was adjusted to 0.16!M with water.

晶析槽の温度を5°Cに設定し、0.09f2/hの循
環流速で濾液循環を行い反応を開始した。反応開始2,
3.および12時時間−フェニルピルビン酸ナトリウム
をそれぞれ0.031mo(ずつ追加し、反応を続けた
ところ、25.5時間後フェニルピルビン酸は完全にな
くなり、晶析槽にL−フェニルアラニンの結晶が蓄積し
た。
The temperature of the crystallization tank was set at 5°C, and the filtrate was circulated at a circulation flow rate of 0.09 f2/h to start the reaction. Reaction start 2,
3. and 12 hours - 0.031 mo (0.031 mo) of sodium phenylpyruvate was added to each, and the reaction was continued. After 25.5 hours, phenylpyruvic acid completely disappeared, and crystals of L-phenylalanine were accumulated in the crystallization tank. .

フェニルピルビン酸のし一フェニルアラニンへの転換率
は92.4%で、そのうち65.5%が固相に、そして
残りの34,5%が液相に存在していた。
The conversion rate of phenylpyruvic acid to monophenylalanine was 92.4%, of which 65.5% was present in the solid phase and the remaining 34.5% in the liquid phase.

上記の実施例2で使用した生体触媒の固定化IH品を用
い、従来の晶析槽を備えた装置、およびカラムを用いる
連続系で以下の実験例に従って反応を行い、その結果を
比較した。
Using the immobilized IH product of the biocatalyst used in Example 2 above, a reaction was carried out according to the following experimental example in an apparatus equipped with a conventional crystallization tank and a continuous system using a column, and the results were compared.

実験例1 (a)晶析槽を備えた装置を用いる方法実施例2− (
2)で調製した濃縮微生物細胞をに一カラギーナンゲル
で包括した固定化パラコツカスデニトリフィカンスを用
いてL−フェニルアラニンのけん副反応を行った。
Experimental Example 1 (a) Method using an apparatus equipped with a crystallization tank Example 2 - (
A side reaction of L-phenylalanine was carried out using immobilized Paracoccus denitrificans prepared by enclosing the concentrated microbial cells prepared in 2) in a carrageenan gel.

(1)装置 実施例2の装置におけるUF膜に代えて、目皿式撹拌捨
型反応器(実効容積0.13のと実効容積0、32 E
Mの晶析槽を閉回路で連結(連結様式は第2図参照)し
たものを用いた。
(1) In place of the UF membrane in the device of device example 2, a perforated stirred waste type reactor (with an effective volume of 0.13 and an effective volume of 0.32 E
The crystallization tanks of M were connected in a closed circuit (see Figure 2 for the connection style).

(2)固定化生体触媒 固定化微生物細胞は次の如(調製した。(2) Immobilized biocatalyst Immobilized microbial cells were prepared as follows.

まず上記濃縮固定化微生物細胞80gに生理食塩水を加
えて全量を0.16ρとして40°Cに保温した。
First, physiological saline was added to 80 g of the concentrated and immobilized microbial cells to make the total volume 0.16 ρ and kept at 40°C.

これと別にに一カラギーナン12.5yを含んだ45°
Cの溶液01207t2を用意し、両者を混合してIO
’Cに冷却してゲル化した。
Apart from this, 45° contains 12.5y of carrageenan.
Prepare solution 01207t2 of C, mix both and IO
It was cooled to 'C and gelled.

得られたゲルの塊を2%塩化カリウム溶液に浸せきし、
10°C以下で一夜放置してから、平均粒径3m/mφ
に成形し、成形品を2%塩化カリウム溶液で洗浄して標
品とした。
The resulting gel mass was soaked in a 2% potassium chloride solution,
After leaving it at 10°C or less overnight, the average particle size was 3m/mφ.
The molded product was washed with a 2% potassium chloride solution to prepare a standard sample.

(3)操作 上記(2)で調製した標品559を反応槽に充填シ、同
時に晶析槽には0.1mo(!のフェニルピルビン酸ナ
トリウム、0.45moCのし一アスパラギン酸アンモ
ニウム、4.5 X 10−5moQビリドキサル5°
−リン酸を充填し、全量を0.315Q(pH8,0)
とした。
(3) Operation Fill the reaction tank with the sample 559 prepared in (2) above, and at the same time fill the crystallization tank with 0.1 mo(!) sodium phenylpyruvate, 0.45 moC monoammonium aspartate, and 4. 5 X 10-5moQ pyridoxal 5°
- Fill with phosphoric acid and make the total amount 0.315Q (pH 8,0)
And so.

反応槽の温度を30’C1晶折槽の温度を5°Cに調節
して濾液を1.16c/hの流速で循環し反応を開始し
た。
The temperature of the reaction tank was adjusted to 5°C, and the filtrate was circulated at a flow rate of 1.16 c/h to start the reaction.

反応開始5,5.30.5.47.5、および55.5
時F’l目にフェニルピルビン酸ナトリウムを0.05
moCずつ加えて反応を続けたところ、95時間[]に
フェニルピルビン酸は完全になくなり、晶析槽にはL−
フェニルアラニンの結晶が蓄積した。
Reaction initiation 5, 5.30.5.47.5, and 55.5
0.05 sodium phenylpyruvate in the F'l eye
When the reaction was continued by adding moC one by one, phenylpyruvic acid completely disappeared in 95 hours, and L-
Phenylalanine crystals accumulated.

この場合、フェニルピルビン酸のし一フェニルアラニン
への転換率は71.2%で、生成したI2−フェニルア
ラニンのうち、65.3%か固相に、残りの34.7%
力冒夜相に存在した。
In this case, the conversion rate of phenylpyruvic acid to I2-phenylalanine was 71.2%, and of the produced I2-phenylalanine, 65.3% was in the solid phase, and the remaining 34.7% was in the solid phase.
It existed during the power attack.

(b)連続系による方法 上記(a)−(2)で調製した固定化微生物細胞を用い
、連続系でL−フェニルアラニンの生産を行った。
(b) Continuous system method L-phenylalanine was produced in a continuous system using the immobilized microbial cells prepared in (a)-(2) above.

(1)装置および操作 使用した反応器は直径3cmφ、高さ25cmの外浴付
円筒カラムで、この中に固定化微生物細胞0゜0685
&充填(充填率0.585)L、た。
(1) Apparatus and operation The reactor used was a cylindrical column with an external bath of 3 cm in diameter and 25 cm in height.
& Filling (filling rate 0.585) L, ta.

カラムを30’Cに制御し、その中ヘフェニルピルヒン
酸ナトリウム0.25M、L−アスパラキン酸アンモニ
ウム0.325M、ピリドキサル5リン酸10−’Mの
基質溶液(pi(8,0)をポンプによって連続的に供
給した。
The column was controlled at 30'C, and a substrate solution (pi(8,0)) of sodium hephenylpyruphate 0.25M, ammonium L-aspartate 0.325M, and pyridoxal pentaphosphate 10'M was added in the column. Continuously fed by pump.

流速か0.0076り/h(平均滞留時間9h)の時、
カラム出口のフェニルピルビン酸の濃度はOであり、L
−フェニルアラニンの濃度は0.197Mであった。こ
の値から、フェニルピルビン酸のしフェニルアラニンへ
の転換率は788%ト計算される。
When the flow rate is 0.0076 r/h (average residence time 9 h),
The concentration of phenylpyruvic acid at the column outlet is O and L
- The concentration of phenylalanine was 0.197M. From this value, the conversion rate of phenylpyruvic acid to phenylalanine is calculated to be 788%.

以上の実施例2および実験例(a)および(b)の反応
で実際に蓄積したし一フェニルアラニンの濃度と、微生
物細胞力たりのし一フェニルアラニンの生産性をまとめ
ると第1表の如くとなった。
Table 1 summarizes the concentration of phenylalanine actually accumulated in the reactions of Example 2 and Experimental Examples (a) and (b) above, and the productivity of phenylalanine per microbial cell power. Ta.

実施例2  0.721     3.55X 10−
’実験例(a)  0.470     1.97x 
to−’実験例(b)  0.197     172
X10−’−ヒ記表より明らかな如く、本発明方法によ
れば従来の晶析槽を併置した固定化生体触媒反応器や、
カラム型反応器による生産法よりも、高濃度のしフェニ
ルアラニンを蓄積でき、生産性が向上される。
Example 2 0.721 3.55X 10-
'Experimental example (a) 0.470 1.97x
to-' Experimental example (b) 0.197 172
As is clear from the table X10-'-H, according to the method of the present invention, an immobilized biocatalyst reactor juxtaposed with a conventional crystallization tank,
Compared to the production method using a column reactor, a higher concentration of phenylalanine can be accumulated and productivity is improved.

[発明の効果] 上記の如く、本発明方法によれば、簡単な装置と操作に
より、生体触媒が高活性を保った状態で、高濃度の基質
の存在下、生成物を高濃度に製造し、蓄積させることか
できる。従って、基質および生体触媒を無駄なく利用し
、効率よく反応を行うことができる。
[Effects of the Invention] As described above, according to the method of the present invention, a product can be produced at a high concentration in the presence of a high concentration of substrate while the biocatalyst maintains high activity using a simple device and operation. , can be accumulated. Therefore, the substrate and the biocatalyst can be used without waste, and the reaction can be carried out efficiently.

【図面の簡単な説明】[Brief explanation of the drawing]

装 第1図は本発明の実施例を示す置の概略ブロック図、第
2図は他の実施例を示す装置の概略ブローク図である。 図中、(1)は反応槽、(2)はUF膜、(3)は晶析
槽、(4)は濾過器、(5)〜(7)はポンプ、(8)
および(9)は撹拌機、(10)、(10″)および(
10”)は送液ライン。
FIG. 1 is a schematic block diagram of the device showing an embodiment of the present invention, and FIG. 2 is a schematic block diagram of the device showing another embodiment. In the figure, (1) is a reaction tank, (2) is a UF membrane, (3) is a crystallization tank, (4) is a filter, (5) to (7) are pumps, and (8)
and (9) are stirrers, (10), (10″) and (
10”) is the liquid supply line.

Claims (1)

【特許請求の範囲】 1、基質と生体触媒とを反応させて有用物質を製造する
方法において、反応混合物を限外濾過膜に通して生成物
に富む濾液を分離し、該濾液から生成物を析出、蓄積さ
せることを特徴とする方法。 2、限外濾過膜を併置する反応槽からなる反応部と、温
度調節機構を備えた晶析槽に濾過器を併置してなる晶析
部とで構成された装置を用い、反応混合物を反応槽から
限外濾過膜に循環させて生成物に富む濾液を得、該濾液
を生成物の析出温度に設定した晶析槽に送って生成物を
析出させ、濾過器で分離した後、その母液を反応槽に循
環させることからなる請求項1に記載の方法。 3、生体触媒がトランスアミナーゼであり、基質がフェ
ニルピルビン酸およびL−アスパラギン酸であり、生成
物がL−フェニルアラニンである請求項1または2に記
載の方法。 4、反応槽および生体触媒を透過させない限外濾過膜か
らなる反応部と、温度調節機構を有する晶析槽および濾
過器からなる晶析部とを相互に連結してなることを特徴
とする生体触媒反応用の装置。
[Claims] 1. In a method for producing a useful substance by reacting a substrate and a biocatalyst, the reaction mixture is passed through an ultrafiltration membrane to separate a product-rich filtrate, and the product is extracted from the filtrate. A method characterized by precipitation and accumulation. 2. The reaction mixture is reacted using an apparatus consisting of a reaction section consisting of a reaction tank in which an ultrafiltration membrane is placed side-by-side, and a crystallization section consisting of a crystallization tank equipped with a temperature control mechanism and a filter placed side-by-side. The product is circulated from the tank through an ultrafiltration membrane to obtain a product-rich filtrate, and the filtrate is sent to a crystallization tank set at the precipitation temperature of the product to precipitate the product. After separation with a filter, the mother liquor is 2. A method according to claim 1, which comprises circulating the water into the reaction vessel. 3. The method according to claim 1 or 2, wherein the biocatalyst is a transaminase, the substrates are phenylpyruvate and L-aspartic acid, and the product is L-phenylalanine. 4. A living organism characterized by interconnecting a reaction section consisting of a reaction tank and an ultrafiltration membrane that does not allow the biocatalyst to pass therethrough, and a crystallization section consisting of a crystallization tank and a filter having a temperature control mechanism. Equipment for catalytic reactions.
JP63194010A 1988-08-03 1988-08-03 Reaction method using biocatalyst and reaction apparatus thereof Expired - Fee Related JP2520155B2 (en)

Priority Applications (1)

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JP63194010A JP2520155B2 (en) 1988-08-03 1988-08-03 Reaction method using biocatalyst and reaction apparatus thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP63194010A JP2520155B2 (en) 1988-08-03 1988-08-03 Reaction method using biocatalyst and reaction apparatus thereof

Publications (2)

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JPH0242992A true JPH0242992A (en) 1990-02-13
JP2520155B2 JP2520155B2 (en) 1996-07-31

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04335890A (en) * 1991-05-08 1992-11-24 Tanabe Seiyaku Co Ltd Reaction process using immobilized biocatalyst and apparatus therefor
WO2008022852A1 (en) * 2006-08-25 2008-02-28 Dsm Ip Assets B.V. Process for in situ crystallisation of a product in a bioconversion process
WO2011005069A1 (en) * 2009-07-06 2011-01-13 Universiti Sains Malaysia A system for producing l-homophenylalanine and a process for producing l-homophenylalanine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59130180A (en) * 1982-11-11 1984-07-26 韓国科学技術院 Recirculatory ultrafiltration inducing method in continuous diaphragm biochemical reactor
JPS615789A (en) * 1984-06-15 1986-01-11 Tanabe Seiyaku Co Ltd Process for reaction by immobilized biocatalyst
JPS6188872A (en) * 1984-10-09 1986-05-07 Snow Brand Milk Prod Co Ltd Method and apparatus for continuous cultivation at high concentration
JPS62259576A (en) * 1986-05-06 1987-11-11 Toshiba Corp Fermentation apparatus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59130180A (en) * 1982-11-11 1984-07-26 韓国科学技術院 Recirculatory ultrafiltration inducing method in continuous diaphragm biochemical reactor
JPS615789A (en) * 1984-06-15 1986-01-11 Tanabe Seiyaku Co Ltd Process for reaction by immobilized biocatalyst
JPS6188872A (en) * 1984-10-09 1986-05-07 Snow Brand Milk Prod Co Ltd Method and apparatus for continuous cultivation at high concentration
JPS62259576A (en) * 1986-05-06 1987-11-11 Toshiba Corp Fermentation apparatus

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04335890A (en) * 1991-05-08 1992-11-24 Tanabe Seiyaku Co Ltd Reaction process using immobilized biocatalyst and apparatus therefor
WO2008022852A1 (en) * 2006-08-25 2008-02-28 Dsm Ip Assets B.V. Process for in situ crystallisation of a product in a bioconversion process
WO2011005069A1 (en) * 2009-07-06 2011-01-13 Universiti Sains Malaysia A system for producing l-homophenylalanine and a process for producing l-homophenylalanine
EP2451566A4 (en) * 2009-07-06 2017-10-25 Universiti Sains Malaysia A system for producing l-homophenylalanine and a process for producing l-homophenylalanine

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