JPH02145537A - Purification of cis, cis-muconic acid - Google Patents
Purification of cis, cis-muconic acidInfo
- Publication number
- JPH02145537A JPH02145537A JP29958788A JP29958788A JPH02145537A JP H02145537 A JPH02145537 A JP H02145537A JP 29958788 A JP29958788 A JP 29958788A JP 29958788 A JP29958788 A JP 29958788A JP H02145537 A JPH02145537 A JP H02145537A
- Authority
- JP
- Japan
- Prior art keywords
- cis
- muconic acid
- ion exchange
- acid
- solution
- 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
- TXXHDPDFNKHHGW-CCAGOZQPSA-N cis,cis-muconic acid Chemical compound OC(=O)\C=C/C=C\C(O)=O TXXHDPDFNKHHGW-CCAGOZQPSA-N 0.000 title claims abstract description 69
- TXXHDPDFNKHHGW-UHFFFAOYSA-N (2E,4E)-2,4-hexadienedioic acid Natural products OC(=O)C=CC=CC(O)=O TXXHDPDFNKHHGW-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000000746 purification Methods 0.000 title description 4
- 239000011347 resin Substances 0.000 claims abstract description 29
- 229920005989 resin Polymers 0.000 claims abstract description 29
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 20
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 20
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000001179 sorption measurement Methods 0.000 claims abstract description 11
- 230000002378 acidificating effect Effects 0.000 claims abstract description 10
- 239000002253 acid Substances 0.000 claims abstract description 5
- 239000012670 alkaline solution Substances 0.000 claims abstract description 4
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 abstract description 24
- 239000000243 solution Substances 0.000 abstract description 23
- 239000005711 Benzoic acid Substances 0.000 abstract description 12
- 235000010233 benzoic acid Nutrition 0.000 abstract description 12
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 239000002994 raw material Substances 0.000 abstract description 8
- 239000012535 impurity Substances 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000004952 Polyamide Substances 0.000 abstract description 2
- 229920002647 polyamide Polymers 0.000 abstract description 2
- 229920000728 polyester Polymers 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 238000005137 deposition process Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 229920001429 chelating resin Polymers 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- TXXHDPDFNKHHGW-HSFFGMMNSA-N cis,trans-muconic acid Chemical compound OC(=O)\C=C\C=C/C(O)=O TXXHDPDFNKHHGW-HSFFGMMNSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- 241000186063 Arthrobacter Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- RRKODOZNUZCUBN-CCAGOZQPSA-N (1z,3z)-cycloocta-1,3-diene Chemical class C1CC\C=C/C=C\C1 RRKODOZNUZCUBN-CCAGOZQPSA-N 0.000 description 1
- 241000186073 Arthrobacter sp. Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- OGGXGZAMXPVRFZ-UHFFFAOYSA-N dimethylarsinic acid Chemical compound C[As](C)(O)=O OGGXGZAMXPVRFZ-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- QXJQHYBHAIHNGG-UHFFFAOYSA-N trimethylolethane Chemical compound OCC(C)(CO)CO QXJQHYBHAIHNGG-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、シス、シス−ムコン酸の新規な精製法に関す
る。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a novel method for purifying cis, cis-muconic acid.
さらに詳しくは、原料の安息香酸を含む反応液に微生物
菌体による酸化反応を施し、得られたシス、シス−ムコ
ン酸を単離精製する方法に関する。More specifically, the present invention relates to a method of subjecting a reaction solution containing benzoic acid as a raw material to an oxidation reaction using microbial cells, and isolating and purifying the obtained cis, cis-muconic acid.
（従来の技術及び発明が解決しようとする問題点）シス
、シス−ムコン酸は、共役二重結合を有する二塩基性カ
ルボン酸の一つであり、ポリアミド、ポリエステル等の
各種ポリマー原料、あるいはトリメット酸代替物としシ
クロオクタジエン系化合物の原料等の各種有機合成薬品
原料としての利用が期待される化合物である。(Prior art and problems to be solved by the invention) Cis, cis-muconic acid is one of the dibasic carboxylic acids having a conjugated double bond, and is used as a raw material for various polymers such as polyamide and polyester, or trimet. It is a compound that is expected to be used as an acid substitute and as a raw material for various organic synthetic drugs such as a raw material for cyclooctadiene compounds.
シス、シス−ムコン酸を化学的に合成する方法として、
銅触媒を用いてカテコールを酸化する方法等があるが、
触媒の回収が困難である等の問題点があり、工業的な応
用には至らなかった。As a method for chemically synthesizing cis, cis-muconic acid,
There are methods to oxidize catechol using a copper catalyst, etc.
There were problems such as difficulty in recovering the catalyst, and industrial application was not possible.
また、微生物触媒による製造法として、コリネバクテリ
ウム グルタミカムによる方法（発酵工学雑誌、第５５
巻 第２号、９５−９７．１９７７）、シュードモナス
プチダによる方法（特開昭５８−４００９２号公報）
等が公知ではあるが、これらによるシス、シス−ムコン
酸の生産性は最大で５ｇ／ｌ程度であり、かつ原料（安
息香酸、あるいはトルエン）からの変換率もｌＯ％程度
と低く、工業的に実施するには不充分である。In addition, as a production method using a microbial catalyst, a method using Corynebacterium glutamicum (Fermentation Engineering Journal, No. 55)
Volume No. 2, 95-97.1977), method using Pseudomonas putida (Japanese Patent Application Laid-Open No. 1983-40092)
However, the maximum productivity of cis, cis-muconic acid by these methods is about 5 g/l, and the conversion rate from the raw material (benzoic acid or toluene) is as low as about 10%, making it difficult for industrial use. It is not sufficient to implement it.
このような状況において、本発明者らは先にアルスルバ
クター属の細菌が特に良好に安息香酸からシス、シス−
ムコン酸に変換することを見出し、この菌を用いた工業
プロセスを提案した（特開昭６２−２９９９２号公報）
。Under these circumstances, the present inventors have previously demonstrated that bacteria of the genus Arsulbacter are particularly effective at recovering from benzoic acid in cis and cis-
He discovered that it can be converted to muconic acid and proposed an industrial process using this bacterium (Japanese Patent Application Laid-Open No. 62-29992).
.
この方法によると、安息香酸を原料としてシス。According to this method, benzoic acid is used as a raw material to produce cis.
シス−ムコン酸は最高４０ ｇ／ｆまで選択的に変換蓄
積し得る。ところが、シス、シス−ムコン酸を含有する
培養液中には、菌の生育に伴って生じる老廃物等から成
る多量の夾雑物が含有されているため、シス、シス−ム
コン酸だけを効率的に分離精製して得ることは困難であ
る。通常は、このような有機酸の分離を行なう場合には
、塩基性樹脂に吸脱着させることにより濃縮・精製の目
的が達せられるが、シス、シス−ムコン酸の場合は培養
物をそのまま塩基性樹脂に通導させても吸着性が悪く、
精製度が上がらないという問題があった。Cis-muconic acid can be selectively converted and accumulated up to 40 g/f. However, since the culture solution containing cis, cis-muconic acid contains a large amount of impurities consisting of waste products generated with the growth of bacteria, it is difficult to efficiently extract only cis, cis-muconic acid. It is difficult to separate and purify it. Normally, when separating such organic acids, the purpose of concentration and purification is achieved by adsorbing and desorbing them to a basic resin. Even if the resin is conductive, the adsorption properties are poor.
There was a problem that the degree of purification did not increase.
（問題点を解決するための手段）
そこで、本発明者らは、培養物からシス、シス−ムコン
酸を良好に精製し得る方法について鋭意検討を重ねた結
果、培養物をマクロポーラス非イオン性吸着樹脂に通導
することによって、未反応の安息香酸及び着色性の夾雑
物を除去し、さらに強塩基性のイオン交換樹脂に通導し
て前処理後、弱塩基性イオン交換樹脂に吸着させ、次い
でＮａＯＨ等のアルカリ性溶液による溶出を行い、塩酸
等の酸で酸析を行なうことにより高純度のシス。(Means for Solving the Problems) Therefore, the present inventors have conducted intensive studies on a method for satisfactorily purifying cis, cis-muconic acid from a culture. Unreacted benzoic acid and colored impurities are removed by passing through an adsorption resin, and after pretreatment by passing through a strongly basic ion exchange resin, the mixture is adsorbed onto a weakly basic ion exchange resin. Then, high purity cis is obtained by elution with an alkaline solution such as NaOH and acid precipitation with an acid such as hydrochloric acid.
シス−ムコン酸を得ることが可能であることを見出し、
本発明を完成するに至った。discovered that it is possible to obtain cis-muconic acid,
The present invention has now been completed.
以下、本発明を説明するに、本発明において精製の対象
となる反応液としてはシス、シス−ムコン酸に選択的に
変換する能力を有するならばいかなる菌体、あるいはそ
の酵素による培養物でも良く、たとえば特開昭６２−２
９９９２号公報に記載のアルスロバクタ−属の菌株 微
工研条寄第１０３６号（旧微工研菌寄第７９５３号）に
よるもの、あるいは微工研条寄第１０３５号（旧微工研
菌寄第７９５２号）によるもの等が挙げられる。Hereinafter, to explain the present invention, the reaction solution to be purified in the present invention may be any bacterial cell or its enzyme culture as long as it has the ability to selectively convert into cis, cis-muconic acid. , for example, JP-A-62-2
Strains of the genus Arthrobacter described in Publication No. 9992; Strains of the genus Arthrobacter described in Publication No. 1036 (formerly No. 7953); No. 7952).
例えば、この菌株を用い、安息香酸を原料としてシス、
シス−ムコン酸を含む培養物を得る方法は以下の通りで
ある。For example, using this strain and using benzoic acid as a raw material, cis,
The method for obtaining a culture containing cis-muconic acid is as follows.
ペプトン、酵母エキス、塩化ナトリウム等より成る培養
液を仕込んだ発酵槽、あるいはフラスコにかかる菌株を
接種し、通気撹拌しながら３０°Ｃで１８時間培養する
。しかる後、多量の菌体を含むその培養物に、安息香酸
、ペプトン及び酵母エキスを含む反応液を逐次添加しな
がら、さらに２日間培養・反応を行なうと、安息香酸は
シス、シス−ムコン酸にほぼ全て変換され、最高４０
ｇ／ｌまで蓄積される。The strain is inoculated into a fermenter or flask containing a culture solution containing peptone, yeast extract, sodium chloride, etc., and cultured at 30°C for 18 hours with aeration and stirring. Thereafter, a reaction solution containing benzoic acid, peptone, and yeast extract was successively added to the culture containing a large amount of bacterial cells, and the culture and reaction were continued for two more days. Almost all are converted to , up to 40
It accumulates up to g/l.
このようにして得た培養物中には、ペプトン、酵母エキ
ス等の代謝によって生じた夾雑物を含んでおり、反応の
経過によっては未反応の安息香酸も含まれるが、本発明
における分離・精製法においては何ら差支えない。The culture obtained in this way contains impurities generated by metabolism such as peptone and yeast extract, and depending on the progress of the reaction, unreacted benzoic acid may also be included. There is no problem in law.
以下、本発明におけるシス、シス−ムコン酸の分離精製
方法につき、詳細に説明する。Hereinafter, the method for separating and purifying cis, cis-muconic acid in the present invention will be explained in detail.
本発明における精製工程では、まず前処理として上記培
養物を遠心分離等により除菌したシス。In the purification step of the present invention, first, as a pretreatment, the culture is sterilized by centrifugation or the like.
シス−ムコン酸を含有する溶液をマクロポーラス非イオ
ン性吸着樹脂塔へ通導し、夾雑物及び安息香酸を除去す
る。この方法で使用されるマクロポーラス非イオン性吸
着樹脂としては、スチレンとジビニルベンゼンとの共重
合体をその樹脂母体とし、かつ半径分布が１００〜５０
０人の細孔を有するものが望ましいが、これに限定され
るものではない。かかるマクロポーラス非イオン性吸着
樹脂の具体例としては、アンバーライト ＸＡＤ−１、
同ＸＡＤ−２、同ＸＡＤ−４、同ＸＡＤ−７、同ＸＡＤ
−８、同ＸＡＤ−９、同ＸＡＤ−１１、同 ＸＡＤ−１
２（商標：ローム・アンド・ハース社製）、ダイヤイオ
ン ＨＰｌＯ１同ＨＰ−２０、同ＨＰ−３０，同ＨＰ−
４０、同 ＨＰ−５０（商標：三菱化成株式会社製）、
イマクテ４ ５ｙｎ−４２、同 ５ｙｎ−４４、同 ５
ｙｎ−４６（商標ｓＩＭＡＣＴＩ社製）等が挙げられる
。かかる樹脂にて前処理を行なうことのできる反応液の
容量は、樹脂塔容量のほぼ１００倍であった。なお、か
かる樹脂の再生は、例えばアセトン、含水アセトン、メ
タノール等の有機溶媒で樹脂を洗浄することにより、容
易に行うことができる。The solution containing cis-muconic acid is passed through a macroporous nonionic adsorption resin column to remove impurities and benzoic acid. The macroporous nonionic adsorption resin used in this method uses a copolymer of styrene and divinylbenzene as its base resin, and has a radius distribution of 100 to 50.
A material having 0 pores is desirable, but is not limited thereto. Specific examples of such macroporous nonionic adsorption resins include Amberlite XAD-1,
Same XAD-2, Same XAD-4, Same XAD-7, Same XAD
-8, XAD-9, XAD-11, XAD-1
2 (trademark: manufactured by Rohm and Haas), Diaion HPlO1 HP-20, HP-30, HP-
40, HP-50 (trademark: manufactured by Mitsubishi Kasei Corporation),
Imakute 4 5yn-42, Imakute 5yn-44, Imakute 5
Examples include yn-46 (trademark manufactured by sIMACTI). The capacity of the reaction liquid that could be pretreated with such a resin was approximately 100 times the capacity of the resin column. Note that such resin regeneration can be easily performed by washing the resin with an organic solvent such as acetone, hydrated acetone, or methanol.
次の工程として、本発明においては最終工程での弱塩基
性イオン交換樹脂へのシス、シス−ムコン酸の吸着率を
高めるための、強酸性イオン交換樹脂塔への通導が行な
われる。この工程での使用に適した強酸性イオン交換樹
脂の具体例としては、アンバーライト ｌＲ１１２、同
ｌＲ１２２、同 ｌＲ２００（商標：ローム・アンド
・ハース社製）、ダイヤイオン ５Ｋ１０２、同 ５Ｋ
ＩｌＯ１同 ＰＫ２０Ｂ、同 ＨＰＫ２５ （商標二三
菱化成株式会社製）、ダウエックス ５０ＷＸ２、同
５０ＷＸ８、同 ５０ＷＸ１０（商標：ダウ ケミカル
社製）等のイオン型がＨ（水素）型のものが挙げられる
。かかる強酸性イオン交換樹脂に、前述の吸着樹脂処理
を施した溶液を通導すると、液のｐＨが低下する。シス
、シス−ムコン酸は、ｐＨ５以下の状態では水に対する
溶解度が１ｇ／２と低いため、その析出を避けるために
、本工程の前に予め溶液のシス、シス−ムコン酸の濃度
をＩｇ／４２以下となるように水で希釈してお（ことが
好ましい。本工程によるイオン交換の終点は、溶出液の
ｐＨが３になる時点であり、通導可能な反応液の容量は
、樹脂塔容量の４倍に相当した。使用後の強酸性イオン
交換樹脂は、過剰量の塩酸水溶液を通導することにより
、容易に再生される。As the next step, in the present invention, in order to increase the adsorption rate of cis, cis-muconic acid to the weakly basic ion exchange resin in the final step, conduction is conducted to a strongly acidic ion exchange resin column. Specific examples of strongly acidic ion exchange resins suitable for use in this process include Amberlite 1R112, Amberlite 1R122, Amberlite 1R200 (trademark: manufactured by Rohm and Haas), Diaion 5K102, Amberlite 5K
IlO1 PK20B, HPK25 (trademark 2, manufactured by Mitsubishi Kasei Corporation), DOWEX 50WX2,
Examples include those in which the ion type is H (hydrogen) type, such as 50WX8 and 50WX10 (trademark: manufactured by Dow Chemical Company). When a solution treated with the adsorption resin described above is passed through such a strongly acidic ion exchange resin, the pH of the solution is lowered. Since the solubility of cis, cis-muconic acid in water is as low as 1 g/2 when the pH is below 5, in order to avoid precipitation, the concentration of cis, cis-muconic acid in the solution is adjusted to Ig/2 before this step. 42 or less (preferably. The end point of ion exchange in this step is when the pH of the eluate becomes 3, and the volume of the reaction solution that can be passed through the resin column is This corresponded to four times the capacity.The strongly acidic ion exchange resin after use can be easily regenerated by passing an excess amount of aqueous hydrochloric acid solution through it.
３番目の工程として、弱塩基性イオン交換樹脂によるシ
ス、シス−ムコン酸の吸着・溶出が行なわれる。この工
程での使用に適した弱塩基性イオン交換樹脂の具体例と
しては、アンバーライトＩＲＡ３８、同 ｌＲ４５、同
ＩＲＡ９３（商標：ローム・アンド・ハース社製）、
ダイヤイオンＷＡＩＯ１同ＷＡＬＬ、同ＷＡ２０、同Ｗ
Ａ２１、同 ＷＡ３０（商標二三菱化成株式会社製）等
のイオン型がＯＨ型のものが挙げられる。As the third step, cis, cis-muconic acid is adsorbed and eluted using a weakly basic ion exchange resin. Specific examples of weakly basic ion exchange resins suitable for use in this step include Amberlite IRA38, Amberlite IRA45, Amberlite IRA93 (trademark: manufactured by Rohm and Haas),
Diaion WAIO1 WALL, WA20, W
Examples include those whose ionic type is OH type, such as A21 and WA30 (trademark manufactured by Mitsubishi Kasei Corporation).
本工程では、かかる弱塩基性イオン交換樹脂を充填した
樹脂塔に前述の強酸性イオン交換樹脂による処理を施し
た反応液を通導し、シス、シス−ムコン酸を吸着、次い
で水酸化ナトリウム等のアルカリ性水溶液により溶出す
る。反応後の通導終点は、通過した液のｐＨが６以下と
なる時点であり、通導可能反応液の容積は、樹脂塔容積
の８倍に相当した。吸着されたシス、シス−ムコン酸の
溶出に際しては、溶離液を樹脂塔容積に相当する分を分
画分取し、高速液体クロマトグラフィーによって測定し
たシス、シス−ムコン酸の含有濃度の高い分画を集める
と、効率良（シス、シス−ムコン酸が回収できる。なお
、この溶出工程に際し、樹脂の交換容量に対して過剰の
アルカリ性溶液を通導することにより、樹脂の再生をも
兼ねることができる。In this step, the reaction solution treated with the above-mentioned strong acidic ion exchange resin is passed through a resin tower filled with the weakly basic ion exchange resin, and cis and cis-muconic acids are adsorbed, and then sodium hydroxide, etc. Elutes with alkaline aqueous solution. The end point of conduction after the reaction was the time point when the pH of the liquid that had passed became 6 or less, and the volume of the reaction liquid that could be conducted was equivalent to eight times the volume of the resin column. When eluting the adsorbed cis, cis-muconic acid, the eluent is fractionated in an amount corresponding to the volume of the resin column, and the fraction containing a high concentration of cis, cis-muconic acid is measured by high-performance liquid chromatography. By collecting the fraction, cis, cis-muconic acid can be efficiently recovered.In addition, during this elution process, by passing an alkaline solution in excess of the exchange capacity of the resin, it can also be used to regenerate the resin. Can be done.
これらの樹脂により精製されたシス、シス−ムコン酸溶
液に、濃塩酸を加えてｐＨを３以下にまで低下させてシ
ス、シス−ムコン酸を析出させ、ろ取、水洗の後、乾燥
する。これら一連の操作によって得られるシス、シス−
ムコン酸の回収率は９０％以上であり、異性体への変化
を伴うこともな（、立体異性的にも高純度なシス、シス
−ムコン酸を得ることができる。Concentrated hydrochloric acid is added to the cis, cis-muconic acid solution purified using these resins to lower the pH to 3 or less to precipitate cis, cis-muconic acid, which is filtered, washed with water, and then dried. Cis, cis- obtained by these series of operations
The recovery rate of muconic acid is 90% or more, and there is no change to isomers (cis, cis-muconic acid with high stereoisomerism can be obtained).
（実施例）
次に、実施例により本発明を更に具体的に説明するが、
その要旨を越えない限り、以下の実施例に限定されるも
のではない。(Example) Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist thereof.
実施例 １
安息香酸を基質として、アルスロバクタ−エスピー 微
工研条寄第１０３６号（旧微工研菌寄第７９５３号）に
より、シス、シス−ムコン酸を生成させた反応ろ液（シ
ス、シス−ムコン酸 ｌＯｇ／β、安息香酸 ０．５
ｇ ／ １ 含有）を精製水に希釈して、１０２の溶
液とした。Example 1 A reaction filtrate (cis, cis) in which cis, cis-muconic acid was produced using Arthrobacter sp. -Muconic acid lOg/β, benzoic acid 0.5
g/1) was diluted with purified water to make a solution of 102.
このろ液を、マクロポーラス非イオン性吸着樹脂 ダイ
ヤイオン ＨＰ−２０１００ｍｌ、を充填した内径３０
ｍｍの樹脂塔に ５Ｖ＝１ で通導し、次いで強酸性
イオン交換樹脂 ダイヤイオン５Ｋ１０２（Ｈ型）５０
０ｍｆを充填した内径６０鵬の樹脂塔に ５Ｖ＝１
で通導し、前処理溶液を得た。This filtrate was filled with 100ml of macroporous nonionic adsorption resin Diaion HP-20.
5V = 1 to the resin column of 1 mm, and then a strong acidic ion exchange resin Diaion 5K102 (H type) 50
5V=1 in a resin tower with an inner diameter of 60mm filled with 0mf
A pretreatment solution was obtained.
この前処理溶液を、弱塩基性イオン交換樹脂ダイヤイオ
ン ＷＡ３０（ＯＨ型）２５０ｍｆを充填した内径４０
ｍｍの樹脂塔に ５Ｖ＝１ で通導し、シス、シス−
ムコン酸を吸着させた。吸着後、カラム中の樹脂を２５
０ｍｆの水で洗浄し、次いで水酸化ナトリウム溶液（２
規定）を Ｓ■＝１ で通液して、シス、シス−ムコン
酸を溶離させた。This pretreatment solution was applied to a tube with an inner diameter of 40 mf filled with weakly basic ion exchange resin Diamond WA30 (OH type) 250 mf.
Conducted at 5V=1 to the resin tower of mm, cis, cis-
Muconic acid was adsorbed. After adsorption, the resin in the column was
Wash with 0 mf water and then with sodium hydroxide solution (2
cis, cis-muconic acid was eluted by passing S■=1.
シス、シス−ムコン酸含有分画５００ｎｆ！（溶出率
９５％；シス、シス−ムコンＨ１９ｇ／ｌ含有）に３５
％塩酸を加えてｐＨを２とし、析出物をろ取して５００
ｍｌの水で洗浄し、４０°Ｃの減圧下（約１００ ｍｍ
Ｈｇ）で１２時間乾燥を行い、白色の精製シス、シス−
ムコン酸結晶 ９．１ ｇを得た。Cis, cis-muconic acid-containing fraction 500nf! (elution rate
95%; Contains cis, cis-mucone H19g/l) to 35
% hydrochloric acid was added to adjust the pH to 2, and the precipitate was collected by filtration.
ml of water and dried under reduced pressure at 40°C (approximately 100 mm
Hg) for 12 hours to obtain a white purified cis, cis-
9.1 g of muconic acid crystals were obtained.
得られたシス、シス−ムコン酸の滴定純度は９９．６％
、融点 １９２−１９４°Ｃ１収率は９１％であった。The titration purity of the obtained cis, cis-muconic acid was 99.6%.
, melting point 192-194°C1 yield was 91%.
また、薄層クロマトグラフィー（担体 シリカゲル；溶
媒 クロロホルム：酢酸＝９：ｌ）及び高速液体クロマ
トグラフィー（担体Ｓｈｉｍｐａｋ ＯＤＳ φ４
．６ Ｘ ２５０ ｍｍ ；溶媒 ３０％メタノール（
ｐＨ２，１ニリン酸による））による分析において、安
息香酸及び異性体の存在は認められなかった。（なお、
Ｓ■とは樹脂塔に１分間通導される液量を樹脂塔容積で
割った値を示す）
（発明の効果）
本発明によれば、培養物から良好にシス、シス−ムコン
酸分離・精製することができる。In addition, thin layer chromatography (carrier silica gel; solvent chloroform:acetic acid = 9:l) and high performance liquid chromatography (carrier Shimpak ODS φ4
．． 6 x 250 mm; Solvent 30% methanol (
The presence of benzoic acid and isomers was not observed in the analysis by pH 2.1 dilic acid)). (In addition,
(S■ indicates the value obtained by dividing the amount of liquid passed through the resin tower for one minute by the volume of the resin tower.) (Effects of the Invention) According to the present invention, cis and cis-muconic acids can be successfully separated from the culture. Can be purified.
Claims (1)
ラス非イオン性吸着樹脂及び強酸性イオン交換樹脂に通
導し、次いで弱塩基性イオン交換樹脂に吸着せしめ、ア
ルカリ性溶液によって溶出し、溶出されたシス、シス−
ムコン酸を含有する水溶液よりシス、シス−ムコン酸を
酸で析出させることを特徴とするシス、シス−ムコン酸
の精製方法。(1) A culture containing cis, cis-muconic acid is passed through a macroporous nonionic adsorption resin and a strongly acidic ion exchange resin, then adsorbed on a weakly basic ion exchange resin, and eluted with an alkaline solution. cis, cis-
1. A method for purifying cis, cis-muconic acid, which comprises precipitating cis, cis-muconic acid from an aqueous solution containing muconic acid with an acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29958788A JPH02145537A (en) | 1988-11-29 | 1988-11-29 | Purification of cis, cis-muconic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP29958788A JPH02145537A (en) | 1988-11-29 | 1988-11-29 | Purification of cis, cis-muconic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02145537A true JPH02145537A (en) | 1990-06-05 |
| JPH0312049B2 JPH0312049B2 (en) | 1991-02-19 |
Family
ID=17874563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP29958788A Granted JPH02145537A (en) | 1988-11-29 | 1988-11-29 | Purification of cis, cis-muconic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02145537A (en) |
-
1988
- 1988-11-29 JP JP29958788A patent/JPH02145537A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0312049B2 (en) | 1991-02-19 |
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