JP5295622B2 - Method for producing target compound using bacterial cell catalyst - Google Patents

Method for producing target compound using bacterial cell catalyst Download PDF

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JP5295622B2
JP5295622B2 JP2008114880A JP2008114880A JP5295622B2 JP 5295622 B2 JP5295622 B2 JP 5295622B2 JP 2008114880 A JP2008114880 A JP 2008114880A JP 2008114880 A JP2008114880 A JP 2008114880A JP 5295622 B2 JP5295622 B2 JP 5295622B2
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cell catalyst
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bacterial cell
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JP2009261320A (en
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孝行 小林
耕三 村尾
雅人 竹内
康治 森
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Dia Nitrix Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an objective compound in an aqueous medium using a microbial catalyst while efficiently and sufficiently coagulating the microbial catalyst after the reaction to enable the separation of the catalyst by a simple separation method. <P>SOLUTION: Provided is a method for producing an objective compound by reacting a substrate compound in an aqueous medium in the presence of a microbial catalyst, characterized in that a cationic coagulant having an amidine group is added to the reaction system after the reaction and the coagulated catalyst is separated from the system. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

本発明は、菌体触媒の存在下で基質化合物を反応させて目的化合物を製造する方法に関する。また本発明は、目的化合物の生成後に反応系から菌体触媒を分離する方法に関する。   The present invention relates to a method for producing a target compound by reacting a substrate compound in the presence of a bacterial cell catalyst. The present invention also relates to a method for separating a cell catalyst from a reaction system after production of a target compound.

酵素触媒を利用して基質化合物を反応させ、目的化合物(生成化合物とも言う)を製造する方法は、反応条件が穏和であること、副生物が少なく反応生成物の純度が高いこと、製造プロセスを簡略化できること等の利点があるため、近年、多くの化合物の製造に用いられている。特に、菌体触媒を用いた場合、酵素の活性が安定化すること、及びその後の触媒の分離(除去)の操作性が優れること等の理由から、工業的に多く使用されている。
アミド化合物の製造においても、ニトリル化合物からアミド化合物に変換する酵素、すなわちニトリルヒドラターゼが見出されて以来、該酵素を産生し得る菌体触媒がアミド化合物の製造に広く利用されている。
A method for producing a target compound (also referred to as a product compound) by reacting a substrate compound using an enzyme catalyst is that the reaction conditions are mild, there are few by-products, and the purity of the reaction product is high. In recent years, it has been used for the production of many compounds because of its advantages such as simplification. In particular, when a bacterial cell catalyst is used, it is widely used industrially for reasons such as stabilization of enzyme activity and excellent operability in subsequent separation (removal) of the catalyst.
Also in the manufacture of amide compounds, since the discovery of an enzyme that converts a nitrile compound into an amide compound, that is, nitrile hydratase, a cell catalyst capable of producing the enzyme has been widely used in the manufacture of amide compounds.

菌体触媒を使用したプロセスでは、原料となる基質化合物と、菌体又は菌体処理物とを接触させて、基質化合物を目的化合物に変換するが、従来は、菌体をゲル等に固定化し、原料と接触させる方法が主流であった。しかし近年では、固定化せずに、菌体又は菌体を死滅等の処理したもの(以下、菌体等と言う)をそのまま基質化合物と接触させて、反応を行うプロセスが開発されている。この方法は、菌体の固定化が不要で、反応性も高くなり、プロセスとしては簡略化されるメリットがある。ところが、この場合、菌体等の反応液からの分離が、固定化して用いた場合より困難となる欠点がある。
菌体触媒を反応系(反応液)から分離する方法としては、膜を利用する方法や遠心分離を使用する方法が知られている。また、特定のカチオン系凝集剤と非イオン性凝集剤とを用いて菌体触媒を凝集させて分離することで、より分離効率を向上させる方法も知られている(特許文献1)。
In a process using a cell catalyst, the substrate compound as a raw material is brought into contact with the cell or a treated product of the cell to convert the substrate compound into the target compound. Conventionally, the cell is immobilized on a gel or the like. The mainstream method is to contact the raw material. However, in recent years, a process has been developed in which a reaction is carried out by bringing a bacterial cell or a treated bacterial cell (hereinafter referred to as a bacterial cell or the like) into contact with a substrate compound as it is without immobilization. This method does not require fixation of bacterial cells, increases the reactivity, and has the advantage of being simplified as a process. However, in this case, there is a drawback that the separation from the reaction solution such as the bacterial cells becomes more difficult than the case where it is used after being immobilized.
As a method for separating the cell catalyst from the reaction system (reaction solution), a method using a membrane or a method using centrifugation is known. Also known is a method of further improving the separation efficiency by aggregating and separating the bacterial cell catalyst using a specific cationic flocculant and a nonionic flocculant (Patent Document 1).

特表2003-507032号公報Special table 2003-507032 gazette

しかしながら、膜分離の方法では、菌体を分離するためにサブミクロン以下の小孔径の膜が必要であり、工業的スケールにおいては、多大な膜分離設備が必要となる。また、遠心分離機を使用する場合には、膨大なエネルギーが必要となる。さらに、特許文献1に記載の方法では、菌体触媒の凝集性がまだ不十分であり、生成させた目的化合物に菌体触媒に付着したまま回収されることで収率が低下することになるため、菌体触媒を十分に分離するには膜の併用が必要であった。
そこで、本発明は、菌体触媒を用いて水性媒体中で目的化合物を製造する方法において、反応後の菌体触媒を、簡便な分離操作で分離できるように、効率的かつ十分に凝集させることができる、上記製造方法を提供することを目的とする。
However, the membrane separation method requires a membrane having a small pore size of submicron or less in order to separate cells, and a large amount of membrane separation equipment is required on an industrial scale. In addition, when a centrifuge is used, enormous energy is required. Furthermore, in the method described in Patent Document 1, the aggregation property of the bacterial cell catalyst is still insufficient, and the yield is lowered by recovering the produced target compound while adhering to the bacterial cell catalyst. For this reason, it was necessary to use a membrane together to sufficiently separate the cell catalyst.
Accordingly, the present invention provides a method for producing a target compound in an aqueous medium using a bacterial cell catalyst, and efficiently and sufficiently aggregates the bacterial cell catalyst after the reaction so that it can be separated by a simple separation operation. An object of the present invention is to provide the above production method.

本発明者らは、上記課題を解決するべく鋭意検討を行った。その結果、菌体触媒を用いて目的化合物を製造する方法において、菌体触媒の凝集剤としてアミジン基を有するカチオン系凝集剤を使用することにより、前記課題を解決できることを見出し、本発明を完成するに至った。
すなわち、本発明は以下の通りである。
The present inventors have intensively studied to solve the above problems. As a result, in the method for producing a target compound using a bacterial cell catalyst, it was found that the above problem can be solved by using a cationic flocculant having an amidine group as the flocculant of the bacterial cell catalyst, and the present invention was completed. It came to do.
That is, the present invention is as follows.

(1)水性媒体中、菌体触媒の存在下で基質化合物を反応させて、目的化合物を製造する方法において、アミジン基を有するカチオン系凝集剤を添加して前記触媒を凝集させた後分離することを特徴とする、前記方法。
(2)水性媒体中、菌体触媒の存在下で基質化合物を反応させて目的化合物を生成させた反応系から、該触媒を分離する方法であって、該反応系にアミジン基を有するカチオン系凝集剤を添加した後、前記触媒の凝集体を分離することを特徴とする、前記方法。
(1) In a method for producing a target compound by reacting a substrate compound in an aqueous medium in the presence of a bacterial cell catalyst, the catalyst is aggregated by adding a cationic flocculant having an amidine group and then separated. And said method.
(2) A method for separating a catalyst from a reaction system in which an objective compound is produced by reacting a substrate compound in an aqueous medium in the presence of a bacterial cell catalyst, the cationic system having an amidine group in the reaction system The method, wherein after adding the flocculant, the agglomerates of the catalyst are separated.

上記(1)及び(2)の方法としては、例えば、前記カチオン系凝集剤とアニオン系凝集剤とを併用する方法が挙げられる。この場合、前記カチオン系凝集剤は、前記アニオン系凝集剤を添加した後に添加することが好ましい。
上記(1)及び(2)の方法においては、例えば、前記基質化合物がニトリル化合物であり、かつ前記目的化合物がアミド化合物である方法が挙げられる。ここで、前記ニトリル化合物及びアミド化合物としては、例えば、前記ニトリル化合物がアクリロニトリルであり、かつ前記アミド化合物がアクリルアミドであるか、又は、前記ニトリル化合物が3−シアノピリジンであり、かつ前記アミド化合物がニコチンアミドであることが好ましい。
Examples of the methods (1) and (2) include a method in which the cationic flocculant and the anionic flocculant are used in combination. In this case, the cationic flocculant is preferably added after the anionic flocculant is added.
Examples of the methods (1) and (2) include a method in which the substrate compound is a nitrile compound and the target compound is an amide compound. Here, as the nitrile compound and the amide compound, for example, the nitrile compound is acrylonitrile and the amide compound is acrylamide, or the nitrile compound is 3-cyanopyridine, and the amide compound is Preferably it is nicotinamide.

本発明によれば、目的化合物生成後の反応系からの菌体触媒の分離性を高め、目的化合物の精製過程を容易化することで、結果として当該化合物の収率を高めることができる。本発明は、菌体触媒を用いた工業的スケールでの目的化合物の製造において、菌体触媒の分離(すなわち目的化合物の精製)を低コストかつ省エネルギーで行うことでき、環境への負荷も低減させることができる点で、非常に実用的かつ有用なものである。   According to this invention, the yield of the said compound can be raised as a result by improving the isolation | separation property of the microbial cell catalyst from the reaction system after target compound production | generation, and facilitating the refinement | purification process of a target compound. INDUSTRIAL APPLICABILITY In the production of a target compound on an industrial scale using a bacterial cell catalyst, separation of the bacterial cell catalyst (that is, purification of the target compound) can be performed at low cost and energy saving, and the load on the environment is reduced In that it is very practical and useful.

以下、本発明を詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。   Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

本発明は、前述の通り、水性媒体中、菌体触媒の存在下で基質化合物を反応させて、目的化合物を製造する方法において、該反応後の反応系にアミジン基を有するカチオン系凝集剤を添加して菌体触媒を凝集させた後に当該凝集した菌体触媒(凝集体)を分離することを特徴とする方法である。
また、本発明は、菌体触媒を反応系から効率的かつ十分に分離する方法、すなわち、水性媒体中、菌体触媒の存在下で基質化合物を反応させて目的化合物を生成させた反応系から、菌体触媒を分離する方法であって、当該反応系にアミジン基を有するカチオン系凝集剤を添加した後、菌体触媒の凝集体を分離することを特徴とする方法も含むものである。
As described above, the present invention provides a method for producing a target compound by reacting a substrate compound in an aqueous medium in the presence of a bacterial cell catalyst, and a cationic flocculant having an amidine group in the reaction system after the reaction. The method is characterized by separating the aggregated bacterial cell catalyst (aggregate) after adding and aggregating the bacterial cell catalyst.
The present invention also provides a method for efficiently and sufficiently separating the cell catalyst from the reaction system, that is, from the reaction system in which the substrate compound is reacted in the presence of the cell catalyst in an aqueous medium to produce the target compound. And a method for separating the bacterial cell catalyst, the method comprising separating a bacterial cell aggregate after adding a cationic flocculant having an amidine group to the reaction system.

本発明において菌体触媒とは、目的とする反応を触媒する酵素を含有する(産生し得る)菌体自体又はその処理物が含有される。本発明でいう処理物としては、菌体(生菌体若しくは死滅体)を触媒としての物性や性能や取扱性を向上させるために、薬品等で処理したもの、例えば、死滅化処理したもの(国際公開第01/36592号パンフレット参照)が含有される。ただし、菌体を微細な格子の中に包み込むか又は半透膜性の高分子の皮膜によって被覆する包括固定化処理や、水不溶性のビーズ等の担体に菌体を固定化させる処理をして得られた菌体処理物は、本発明の効果が期待できないため、本発明でいう処理物には含まれない。   In the present invention, the microbial cell catalyst includes the microbial cell itself containing (or capable of producing) an enzyme that catalyzes the target reaction or a processed product thereof. The treated product as used in the present invention is treated with chemicals etc. in order to improve the physical properties, performance and handleability of the bacterial cells (viable cells or dead cells) as a catalyst, for example, killed ( Contains WO 01/36592 pamphlet). However, it is possible to encapsulate the cells in a fine lattice or cover them with a semipermeable membrane, or to fix the cells on a carrier such as water-insoluble beads. The obtained processed bacterial cell is not included in the processed product in the present invention because the effect of the present invention cannot be expected.

本発明でいう水性媒体中で目的化合物を製造する方法とは、溶媒として水を含む液体中で、反応基質となる化合物(基質化合物)を加え、菌体触媒と接触させることで、基質化合物を反応させて目的化合物を製造する方法である。反応槽の様式としては、攪拌槽、充填槽、移動槽、流動槽などのいずれの様式でもよく、工業的に最も適した様式を選択すればよい。また、反応形式についても、連続反応、半連続反応、回分反応のいずれでも構わないが、工業的に目的化合物を大量にかつ効率的に製造しやすい点で、連続反応が好ましい。連続反応とは、反応原料を連続的又は間歇的に供給しながら、反応混合物を全量抜き出すことなく連続的又は間歇的に取り出す反応形式をいう。   The method for producing a target compound in an aqueous medium as referred to in the present invention means that a compound (substrate compound) as a reaction substrate is added in a liquid containing water as a solvent and brought into contact with a bacterial cell catalyst. This is a method for producing a target compound by reacting. The reaction tank may be any of a stirring tank, a filling tank, a moving tank, a fluidized tank, etc., and an industrially most suitable mode may be selected. Also, the reaction format may be any of continuous reaction, semi-continuous reaction, and batch reaction, but continuous reaction is preferred because it is easy to produce the target compound industrially in large quantities and efficiently. The continuous reaction refers to a reaction mode in which a reaction raw material is continuously or intermittently supplied and continuously or intermittently removed without extracting the entire reaction mixture.

本発明でいう目的化合物としては、限定はされず、水性媒体中、菌体触媒の存在下で任意の基質化合物を反応させて(すなわち水和反応させて)製造できるものであればよいが、例えば、ニトリル化合物を基質化合物として製造できるアミド化合物が好ましく、特に、工業的に菌体触媒を用いて水性媒体中で製造されているアクリルアミド及びニコチンアミドが特に好ましい。なお、アクリルアミド及びニコチンアミドを製造する場合に用いる基質化合物(ニトリル化合物)は、それぞれ順に、アクリロニトリル及び3−シアノピリジンである。なお、反応後の目的化合物の含有量(濃度)は、限定はされないが、例えば、1〜60%であることが好ましい。   The target compound as used in the present invention is not limited and may be any compound that can be produced by reacting (i.e., hydrating) an arbitrary substrate compound in an aqueous medium in the presence of a bacterial cell catalyst. For example, an amide compound that can be produced using a nitrile compound as a substrate compound is preferable, and acrylamide and nicotinamide that are industrially produced in an aqueous medium using a bacterial cell catalyst are particularly preferable. In addition, the substrate compound (nitrile compound) used when manufacturing acrylamide and nicotinamide is acrylonitrile and 3-cyanopyridine, respectively. In addition, although content (concentration) of the target compound after reaction is not limited, For example, it is preferable that it is 1 to 60%.

本発明において、反応後の反応系に添加する、菌体触媒を凝集させるための凝集剤としては、アミジン基を有するカチオン系凝集剤を使用することが重要である。アミジン基を有するカチオン系凝集剤としては、具体的には、下記構造式(1)又は(2)のモノマー単位を含んでなるポリマーが好ましく挙げられる。   In the present invention, it is important to use a cationic flocculant having an amidine group as the flocculant added to the reaction system after the reaction to clump the cell catalyst. As the cationic flocculant having an amidine group, specifically, a polymer comprising a monomer unit represented by the following structural formula (1) or (2) is preferably exemplified.

〔上記式(1)及び(2)中、Rとしては、例えば、水素、炭素数1〜3のアルキル基、が挙げられ、中でも水素が好ましく、Rとしては、例えば、水素、炭素数1〜3のアルキル基が挙げられ、中でも水素が好ましい。また、Xとしては、例えば、塩素イオン、硝酸イオン、ギ酸イオン、酢酸イオン、硫酸イオン、等が挙げられ、中でも塩素イオンが好ましい。〕 [In the above formulas (1) and (2), examples of R 1 include hydrogen and an alkyl group having 1 to 3 carbon atoms. Among them, hydrogen is preferable, and R 2 includes, for example, hydrogen and carbon number. 1 to 3 alkyl groups may be mentioned, and hydrogen is particularly preferable. Examples of X include chlorine ions, nitrate ions, formate ions, acetate ions, sulfate ions, and the like. Among these, chlorine ions are preferable. ]

上記構造式(1)又は(2)のモノマー単位を含んでなるポリマーの製造方法は、特に限定はされないが、例えば、アクリロニトリルとビニルアミドとのコポリマー(共重合体)を酸加水分解した後、アミジン環化したものが好ましい。当該ポリマー中に占める上記構造式(1)又は(2)のモノマー単位(アミジンユニット)の割合は、限定はされないが、例えば、5〜80モル%であることが好ましく、10〜70モル%がより好ましい。他に含まれ得るモノマー単位としては、特に限定はされないが、例えば、ニトリル基、アミノ基、アミン塩酸塩、アミド基、ラクタム環、カルボン酸等の側鎖を有するモノマー単位が挙げられる。当該ポリマーの分子量は、限定はされないが、例えば、1Nの食塩水中に0.1g/dLの溶液として25℃で測定した還元粘度が0.1〜10の範囲となる分子量であることが好ましく、より好ましくは、上記還元粘度が0.2〜8の範囲となる分子量である。   The production method of the polymer comprising the monomer unit of the structural formula (1) or (2) is not particularly limited. For example, after acid hydrolysis of a copolymer (copolymer) of acrylonitrile and vinylamide, amidine A cyclized one is preferred. The proportion of the monomer unit (amidine unit) of the structural formula (1) or (2) in the polymer is not limited, but is preferably 5 to 80 mol%, for example, 10 to 70 mol%. More preferred. Although it does not specifically limit as a monomer unit which can be contained elsewhere, For example, the monomer unit which has side chains, such as a nitrile group, an amino group, amine hydrochloride, an amide group, a lactam ring, and carboxylic acid, is mentioned. The molecular weight of the polymer is not limited, but for example, the molecular weight is preferably such that the reduced viscosity measured at 25 ° C. as a 0.1 g / dL solution in 1N saline is in the range of 0.1 to 10, more preferably The molecular weight is such that the reduced viscosity is in the range of 0.2-8.

本発明で用いるアミジン基を有するカチオン系凝集剤のカチオン当量は、限定はされないが、例えば、ポリマー(該凝集剤)1gあたり1〜10meqであることが好ましく、3〜7meq/gがより好ましい。ここで、上記カチオン当量の値は、コロイド滴定法により測定される値である。
本発明における、アミジン基を有するカチオン系凝集剤を添加するとは、菌体触媒が水性媒体中に分散している状態の時に添加すれば良く、目的化合物の生成(製造)の前でも後でも、菌体触媒が凝縮し分離性が向上すれば良い。但し、反応前に添加して凝集した菌体触媒を用いて反応させると、反応性が低下するおそれがあることから、反応後に添加することが好ましい。
The cation equivalent of the cationic flocculant having an amidine group used in the present invention is not limited, but is preferably 1 to 10 meq per 1 g of the polymer (the flocculant), and more preferably 3 to 7 meq / g. Here, the value of the cation equivalent is a value measured by a colloid titration method.
In the present invention, adding a cationic flocculant having an amidine group may be added when the bacterial cell catalyst is dispersed in an aqueous medium, either before or after the production (production) of the target compound. It is sufficient that the cell catalyst is condensed and the separability is improved. However, if the reaction is carried out using a bacterial cell catalyst that has been added and aggregated before the reaction, the reactivity may be lowered.

本発明において、添加するアミジン基を有するカチオン系凝集剤は、通常は、0.05重量%〜0.5重量%の濃度の水溶液で添加することが好ましく、より好ましくは0.1重量%〜0.3重量%である。また、当該凝集剤の反応系への添加量は、反応液の容量に対して1ppm〜1000ppmとなる量であることが好ましく、より好ましくは10ppm〜500ppmである。当該添加後は、さらに攪拌を行うことにより、菌体触媒を凝集させて凝集体を形成させる。
また、本発明においては、アミジン基を有するカチオン系凝集剤と共に、アニオン系凝集剤を併用してもよい。アニオン系凝集剤を併用することにより、さらに機械的分離を行いやすい強固で大きな凝集体を形成させることができる。アニオン系凝集剤を併用する場合は、当該アニオン系凝集剤を反応系に添加して攪拌した後に、上記カチオン系凝集剤を添加して攪拌することが好ましく、強固で大きな凝集体をより容易に形成させることができる。
In the present invention, the cationic flocculant having an amidine group to be added is usually preferably added in an aqueous solution having a concentration of 0.05 wt% to 0.5 wt%, more preferably 0.1 wt% to 0.3 wt%. %. Further, the amount of the flocculant added to the reaction system is preferably 1 ppm to 1000 ppm, more preferably 10 ppm to 500 ppm, based on the volume of the reaction solution. After the addition, the bacterial cell catalyst is aggregated by further stirring to form an aggregate.
In the present invention, an anionic flocculant may be used in combination with a cationic flocculant having an amidine group. By using an anionic flocculant in combination, a strong and large aggregate that is more easily mechanically separated can be formed. When an anionic flocculant is used in combination, it is preferable that the anionic flocculant is added to the reaction system and stirred, and then the cationic flocculant is added and stirred. Can be formed.

アニオン系凝集剤としては、限定はされないが、例えば、アクリル酸重合体、又はアクリルアミドとアクリル酸とのコポリマーが好適である。さらに、アクリルアミド2−メチルプロパンスルホン酸ユニットを含んでもよい。アニオン系凝集剤は、アクリル酸由来のモノマー単位を5〜100モル%含むものであることが好ましく、より好ましくは5〜50モル%である。アニオン系凝集剤の分子量は、4重量%の食塩水に該凝集剤を1重量%溶解させたときの水溶液粘度が、300〜4000mPa・sの範囲となる分子量であることが好ましく、より好ましくは、上記水溶液粘度が1000〜3500mPa・sの範囲となる分子量である。また、アニオン系凝集剤の反応系への添加量は、反応液の容量に対して1ppm〜1000ppmとなる量であることが好ましく、より好ましくは5ppm〜100ppmである。アニオン凝集剤は、水溶液として反応系に添加するが、その濃度は0.01重量%〜0.5重量%であることが好ましく、より好ましくは0.05重量%〜0.3重量%である。
さらに、本発明においては、処理時にゼオライト等の吸着性の粉体を添加することで清澄性を上げることができる。
The anionic flocculant is not limited, but for example, an acrylic acid polymer or a copolymer of acrylamide and acrylic acid is suitable. Furthermore, an acrylamide 2-methylpropanesulfonic acid unit may be included. The anionic flocculant is preferably one containing 5 to 100 mol% of acrylic acid-derived monomer units, more preferably 5 to 50 mol%. The molecular weight of the anionic flocculant is preferably such that the aqueous solution viscosity when the flocculant is dissolved in 4% by weight of saline is in the range of 300 to 4000 mPa · s, more preferably The molecular weight is such that the viscosity of the aqueous solution is in the range of 1000 to 3500 mPa · s. Further, the amount of the anionic flocculant added to the reaction system is preferably an amount of 1 ppm to 1000 ppm, more preferably 5 ppm to 100 ppm, based on the volume of the reaction solution. The anionic flocculant is added to the reaction system as an aqueous solution, and the concentration is preferably 0.01% by weight to 0.5% by weight, more preferably 0.05% by weight to 0.3% by weight.
Furthermore, in the present invention, the clarity can be improved by adding an adsorbent powder such as zeolite during the treatment.

本発明において、菌体触媒の凝集体の分離(除去)方法は、特に限定はされず、例えば、静置沈殿、デカンターを用いた沈殿や遠心力を利用した分離方法、網や膜を利用した分離方法があるが、これも工業的に最も適した形式を選択すればよい。本発明の効果を明確に得るためには、より簡易な方法を採用するのがよく、デカンター分離や網等を利用することが好ましい。   In the present invention, the method for separating (removing) the aggregates of the bacterial cell catalyst is not particularly limited. For example, stationary precipitation, precipitation using a decanter or separation method using centrifugal force, a net or a membrane is used. Although there is a separation method, it is sufficient to select the most industrially suitable format. In order to clearly obtain the effects of the present invention, it is preferable to adopt a simpler method, and it is preferable to use decanter separation, a net, or the like.

本発明の方法においては、目的化合物の生成反応は、反応器を1つ又は複数個用いて実施される。反応器の型式としては、攪拌によって反応器内の流体が混合されるものであればよく、例えば槽型反応器、塔型反応器が挙げられる。攪拌翼の形状は、限定されるものではなく、例えば、パドル、ディスクタービン、プロペラ、ヘリカルリボン、アンカー、ファウドラー、ファンタービン等が挙げられる。
本発明において、以上に述べた各種条件及び方法以外については、基質化合物及び目的化合物の種類、あるいは菌体触媒の種類等に応じて、適宜、公知の条件及び方法を採用して実施することができる。
In the method of the present invention, the production reaction of the target compound is carried out using one or more reactors. Any type of reactor may be used as long as the fluid in the reactor is mixed by stirring, and examples thereof include a tank reactor and a tower reactor. The shape of the stirring blade is not limited, and examples thereof include a paddle, a disk turbine, a propeller, a helical ribbon, an anchor, a fiddler, and a fan turbine.
In the present invention, in addition to the various conditions and methods described above, it may be carried out by appropriately adopting known conditions and methods depending on the types of the substrate compound and the target compound, the type of the bacterial cell catalyst, and the like. it can.

以下、実施例により本発明をさらに具体的に説明するが、本発明はこれらに限定されるものではない。   EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

(1)菌体触媒の調製
ニトリルヒドラターゼ活性を有するロドコッカス・ロドクロウス(Rodococcus rhodochrous)J1株(受託番号:FERM BP-1478として独立行政法人 産業技術総合研究所 特許生物寄託センター(日本国茨城県つくば市東1丁目1番地1中央第6)に1987年9月18日に国際寄託されている)を、グルコース2%、尿素1%、ペプトン0.5%、酵母エキス0.3%、塩化コバルト0.05%(いずれも質量%)を含む培地(pH7.0)により30℃で好気的に培養した。これを遠心分離機及び50mMリン酸緩衝液(pH7.0)を用いて、集菌洗浄し、菌体触媒としての菌体懸濁液(乾燥菌体15質量%)を調製した。
(1) Preparation of bacterial cell catalyst Rhodococcus rhodochrous J1 strain having nitrile hydratase activity (Accession number: FERM BP-1478) National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Tsukuba, Ibaraki, Japan) 1) 1-chome, 1-chome, 1st-century 6)) was deposited internationally on September 18, 1987), glucose 2%, urea 1%, peptone 0.5%, yeast extract 0.3%, cobalt chloride 0.05% (all The medium was aerobically cultured at 30 ° C. in a medium (pH 7.0) containing (mass%). This was collected and washed using a centrifuge and a 50 mM phosphate buffer (pH 7.0) to prepare a cell suspension (dry cell 15% by mass) as a cell catalyst.

(2)3−シアノピリジンからニコチンアミドへの反応
ジャケット付セパラブルフラスコ(内径110mm、高さ150mm)を4槽直列に連結した。第1槽に、14.5%の3−シアノピリジンを溶解させた50mMリン酸緩衝液(pH8.0)を200mL/hrの流速で、上記菌体懸濁液を0.27mL/hrの流速で、それぞれ連続的に添加した。第1槽から第4槽の反応器内の液量を1Lに調整し、第1槽から第4槽の反応温度が、それぞれ順に、23℃、24℃、25℃及び26℃となるようにジャケットの冷却水(10℃)を用いて反応温度を制御した。
3日後、第4槽から流出してくる反応液を、液体クロマトグラフィー(カラム:GLサイエンス社製ODS-80A、溶離液:5%アセトニトリル10mMリン酸緩衝液pH7.0、検出:200nm)で分析したところ、未反応の3−シアノピリジンは検出されず、17.0%のニコチンアミドが検出された。
(2) Reaction from 3-cyanopyridine to nicotinamide Four separable flasks with a jacket (inner diameter 110 mm, height 150 mm) were connected in series. In the first tank, a 50 mM phosphate buffer solution (pH 8.0) in which 14.5% of 3-cyanopyridine was dissolved was flowed at a rate of 200 mL / hr, and the above cell suspension was flowed at a flow rate of 0.27 mL / hr. Added continuously. Adjust the amount of liquid in the reactors from the first tank to the fourth tank to 1 L, and the reaction temperatures from the first tank to the fourth tank are 23 ° C, 24 ° C, 25 ° C, and 26 ° C, respectively. The reaction temperature was controlled using jacket cooling water (10 ° C.).
Three days later, the reaction solution flowing out from the fourth tank was analyzed by liquid chromatography (column: ODS-80A manufactured by GL Sciences, eluent: 5% acetonitrile 10 mM phosphate buffer pH 7.0, detection: 200 nm). As a result, unreacted 3-cyanopyridine was not detected, and 17.0% nicotinamide was detected.

(3)ニコチンアミド水溶液からの菌体触媒の分離
上記反応液300mL(濁度 22.0NTU,pH8.2)を取り、アニオン性凝集剤(アクリルアミド/アクリル酸ソーダ=58/42モル%,ダイヤニトリックス社製,ダイヤフロックAP335B,0.1%水溶液)が、当該反応液に対して10ppmとなるように添加し、100rpmで30秒攪拌した。次いで、アミジン基を有するカチオン系凝集剤(ダイヤニトリックス社製,ダイヤフロックKP7000,0.3%水溶液)を、当該反応液に対して300ppmとなるように添加し、100rpmで30秒攪拌した。その後、60秒静止し、上澄みの濁度、及び沈降した菌体触媒のフロック(凝集体)径を計測した。その結果、フロック径8mm以上、濁度1.95NTUであった。
(3) Separation of bacterial cell catalyst from aqueous nicotinamide solution Take 300 mL of the above reaction solution (turbidity 22.0 NTU, pH 8.2), and anionic flocculant (acrylamide / sodium acrylate = 58/42 mol%, manufactured by Dianitricks) , Diafloc AP335B, 0.1% aqueous solution) was added at 10 ppm to the reaction solution, and stirred at 100 rpm for 30 seconds. Subsequently, a cationic flocculant having an amidine group (manufactured by Dianitics, Diafloc KP7000, 0.3% aqueous solution) was added to 300 ppm with respect to the reaction solution, and stirred at 100 rpm for 30 seconds. Thereafter, the mixture was allowed to stand for 60 seconds, and the turbidity of the supernatant and the floc (aggregate) diameter of the settled bacterial cell catalyst were measured. As a result, the floc diameter was 8 mm or more and the turbidity was 1.95 NTU.

〔比較例1〕
実施例1において、アミジン基を有するカチオン系凝集剤を添加しなかった以外は、同様の操作を行ったところ、十分な菌体のフロック(凝集体)は形成されなかった。
[Comparative Example 1]
In Example 1, except that the cationic flocculant having an amidine group was not added, the same operation was performed, but sufficient fungus flocs (aggregates) were not formed.

実施例1の(1)で調製した菌体懸濁液を用いて、アクリルアミドを製造した。
(1)アクリロニトリルからアクリルアミドへの反応
ジャケット付セパラブルフラスコ(内径110mm、高さ150mm)を7槽直列に連結した。
第1槽目に50mMリン酸緩衝液(pH7.0)を780mL/hrで、アクリロニトリルを175mL/hrの流速で、上記菌体懸濁液を1.1g/hrの流速で、それぞれ連続的に添加し、さらに、第2槽目にはアクリロニトリルのみを175mL/hrの流速で、第3槽にはアクリロニトリルのみを146mL/hrの流速で、第4槽目にはアクリロニトリルのみを87mL/hrの流速で、それぞれ連続的に添加した。第1槽から第7槽の反応液量を1Lに調整し、第1槽から第7槽の反応液温度が、それぞれ順に、21℃、22℃、23℃、24℃、25℃、26℃及び27℃となるようにジャケットの冷却水(10℃)を用いて温度制御した。
2日後、第7槽から流出してくる反応液を、ガスクロマトグラフィー(カラム:Waters社製、PoraPak-PS、1m、180℃、キャリアガス:ヘリウム、検出器:FID)で分析したところ、未反応のアクリロニトリルは検出されず、50.3%のアクリルアミドが検出された。
Acrylamide was produced using the cell suspension prepared in (1) of Example 1.
(1) Reaction from acrylonitrile to acrylamide Seven jacketed separable flasks (inner diameter 110 mm, height 150 mm) were connected in series.
In the first tank, 50 mM phosphate buffer (pH 7.0) was added continuously at 780 mL / hr, acrylonitrile at a flow rate of 175 mL / hr, and the above cell suspension at a flow rate of 1.1 g / hr. Furthermore, in the second tank, only acrylonitrile was flowed at 175 mL / hr, in the third tank, only acrylonitrile was flowed at 146 mL / hr, and in the fourth tank, only acrylonitrile was flowed at 87 mL / hr. Each was added continuously. The amount of reaction liquid in the first tank to the seventh tank is adjusted to 1 L, and the temperature of the reaction liquid in the first tank to the seventh tank is 21 ° C., 22 ° C., 23 ° C., 24 ° C., 25 ° C., 26 ° C., respectively. The temperature was controlled using cooling water (10 ° C.) of the jacket so that the temperature was 27 ° C.
Two days later, the reaction solution flowing out from the seventh tank was analyzed by gas chromatography (column: Waters, PoraPak-PS, 1 m, 180 ° C., carrier gas: helium, detector: FID). No acrylonitrile in the reaction was detected, and 50.3% acrylamide was detected.

(2)アクリルアミド水溶液からの菌体触媒の分離
上記反応液(濁度 21.5NTU,pH8.0)300mLを取り、アニオン性凝集剤(アクリルアミド/アクリル酸ソーダ=58/42モル%,ダイヤニトリックス社製,ダイヤフロックAP335B,0.1%水溶液)が、当該反応液に対して50ppmとなるように添加し(表1参照)、100rpmで30秒攪拌した。次いで、アミジン基を有するカチオン系凝集剤(ダイヤニトリックス社製,ダイヤフロックKP7000,0.3%水溶液)を、当該反応液に対して50ppmとなるように添加し(表1参照)、100rpmで30秒攪拌した。その後、60秒静止し、上澄みの濁度、及び沈降した菌体触媒のフロック(凝集体)径を計測した。その結果を、後述の表1に示す。
(2) Separation of bacterial cell catalyst from acrylamide aqueous solution Take 300 mL of the above reaction solution (turbidity 21.5 NTU, pH 8.0) and use an anionic flocculant (acrylamide / sodium acrylate = 58/42 mol%, manufactured by Daianitrix, (Diafloc AP335B, 0.1% aqueous solution) was added to the reaction solution at 50 ppm (see Table 1) and stirred at 100 rpm for 30 seconds. Next, a cationic flocculant having an amidine group (Dianitricks, Diafloc KP7000, 0.3% aqueous solution) was added to 50 ppm with respect to the reaction solution (see Table 1), and stirred at 100 rpm for 30 seconds. . Thereafter, the mixture was allowed to stand for 60 seconds, and the turbidity of the supernatant and the floc (aggregate) diameter of the settled bacterial cell catalyst were measured. The results are shown in Table 1 described later.

実施例2において、アニオン性凝集剤及びカチオン系凝集剤と併せて、予め、ゼオライト(ユニオン昭和(株)社製,モレキュラーシーブ13Xパウダー)を、前記反応液に対して50ppmとなるように添加した以外は、同様の操作を行って、上澄みの濁度、及び沈降した菌体触媒のフロック(凝集体)径を計測した。その結果を、後述の表1に示す。   In Example 2, together with an anionic flocculant and a cationic flocculant, zeolite (manufactured by Union Showa Co., Ltd., molecular sieve 13X powder) was added in advance so as to be 50 ppm with respect to the reaction solution. Except for the above, the same operation was performed, and the turbidity of the supernatant and the floc (aggregate) diameter of the settled bacterial cell catalyst were measured. The results are shown in Table 1 described later.

〔比較例2〕
実施例2において、ダイヤフロックKP7000の代わりに、ダイヤフロックKP201G(メタクリロイルオキシエチルトリメチルアンモニウムクロリド 100モル%)を反応液に添加した以外は、同様の操作を行ったところ、沈降した菌体触媒のフロック(凝集体)径は1mm以下であり、効率的かつ十分に分離が可能な大きさではなかった。
[Comparative Example 2]
In Example 2, the same procedure was performed except that Diafloc KP201G (methacryloyloxyethyltrimethylammonium chloride 100 mol%) was added to the reaction solution instead of Diafloc KP7000. The (aggregate) diameter was 1 mm or less, and it was not a size that could be separated efficiently and sufficiently.

〔比較例3〕
実施例2において、ダイヤフロックKP7000の代わりに、ダイヤフロックKP204B(メタクリロイルオキシエチルトリメチルアンモニウムクロリド/アクリルアミド=40/60モル%共重合体)を反応液に添加した以外は、同様の操作を行ったところ、沈降した菌体触媒のフロック(凝集体)径は1mm以下であり、効率的かつ十分に分離が可能な大きさではなかった。
[Comparative Example 3]
In Example 2, the same operation was performed except that Diafloc KP204B (methacryloyloxyethyltrimethylammonium chloride / acrylamide = 40/60 mol% copolymer) was added to the reaction solution instead of Diafloc KP7000. The floc (aggregate) diameter of the settled bacterial cell catalyst was 1 mm or less, and it was not a size capable of efficient and sufficient separation.

〔比較例4〕
実施例2において、ダイヤフロックKP7000を反応液に添加しなかった以外は、同様の操作を行ったところ、効率的かつ十分に分離が可能な大きさの菌体触媒のフロック(凝集体)が形成されなかった。
[Comparative Example 4]
In Example 2, the same operation was carried out except that Diafloc KP7000 was not added to the reaction solution. As a result, flocs (aggregates) of the cell catalyst having a size capable of being separated efficiently and sufficiently were formed. Was not.

本発明の方法は、菌体由来の酵素触媒を用いた化学品の製造分野に用いられる。
本発明によれば、目的化合物生成後の反応系からの菌体触媒の分離性を高め、目的化合物の精製過程を容易化することで、結果として当該化合物の収率を高めることができる。本発明は、菌体触媒を用いた工業的スケールでの目的化合物の製造において、菌体触媒の分離(すなわち目的化合物の精製)を低コストかつ省エネルギーで行うことでき、環境への負荷も低減させることができる点で、非常に実用的かつ有用なものである。
The method of the present invention is used in the field of production of chemicals using an enzyme catalyst derived from bacterial cells.
According to this invention, the yield of the said compound can be raised as a result by improving the isolation | separation property of the microbial cell catalyst from the reaction system after target compound production | generation, and facilitating the refinement | purification process of a target compound. INDUSTRIAL APPLICABILITY In the production of a target compound on an industrial scale using a bacterial cell catalyst, separation of the bacterial cell catalyst (that is, purification of the target compound) can be performed at low cost and energy saving, and the load on the environment is also reduced. In that it is very practical and useful.

Claims (4)

水性媒体中、菌体触媒の存在下で基質化合物を反応させて、目的化合物を製造する方法において、アニオン系凝集剤を添加した後に、アミジン基を有するカチオン系凝集剤を添加して前記触媒を凝集させた後分離することを特徴とする、前記方法。 In a method for producing a target compound by reacting a substrate compound in the presence of a bacterial cell catalyst in an aqueous medium, after adding an anionic flocculant , a cationic flocculant having an amidine group is added, and the catalyst The method is characterized in that it is separated after agglomeration. 水性媒体中、菌体触媒の存在下で基質化合物を反応させて目的化合物を生成させた反応系から、該触媒を分離する方法であって、該反応系に、アニオン系凝集剤を添加した後、アミジン基を有するカチオン系凝集剤を添加し、前記触媒の凝集体を分離することを特徴とする、前記方法。 A method for separating a catalyst from a reaction system in which an objective compound is produced by reacting a substrate compound in an aqueous medium in the presence of a cell catalyst, after adding an anionic flocculant to the reaction system with the addition of cationic coagulant having an amidine group, and separating the aggregates of the catalyst, said method. 前記基質化合物がニトリル化合物であり、かつ前記目的化合物がアミド化合物である、請求項1又は2に記載の方法。 The method according to claim 1 or 2 , wherein the substrate compound is a nitrile compound and the target compound is an amide compound. 前記ニトリル化合物がアクリロニトリルであり、かつ前記アミド化合物がアクリルアミドであるか、又は、前記ニトリル化合物が3−シアノピリジンであり、かつ前記アミド化合物がニコチンアミドである、請求項記載の方法。
4. The method according to claim 3 , wherein the nitrile compound is acrylonitrile and the amide compound is acrylamide, or the nitrile compound is 3-cyanopyridine and the amide compound is nicotinamide.
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