JPH0412708B2 - - Google Patents
Info
- Publication number
- JPH0412708B2 JPH0412708B2 JP62028065A JP2806587A JPH0412708B2 JP H0412708 B2 JPH0412708 B2 JP H0412708B2 JP 62028065 A JP62028065 A JP 62028065A JP 2806587 A JP2806587 A JP 2806587A JP H0412708 B2 JPH0412708 B2 JP H0412708B2
- Authority
- JP
- Japan
- Prior art keywords
- muconic acid
- acid
- catechol
- benzoic acid
- producing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- TXXHDPDFNKHHGW-UHFFFAOYSA-N muconic acid Chemical compound OC(=O)C=CC=CC(O)=O TXXHDPDFNKHHGW-UHFFFAOYSA-N 0.000 claims description 46
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 25
- TXXHDPDFNKHHGW-CCAGOZQPSA-N Muconic acid Natural products OC(=O)\C=C/C=C\C(O)=O TXXHDPDFNKHHGW-CCAGOZQPSA-N 0.000 claims description 23
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000005711 Benzoic acid Substances 0.000 claims description 12
- 235000010233 benzoic acid Nutrition 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 241001467578 Microbacterium Species 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 241001517047 Corynebacterium acetoacidophilum Species 0.000 claims description 3
- OMIGHNLMNHATMP-UHFFFAOYSA-N 2-hydroxyethyl prop-2-enoate Chemical compound OCCOC(=O)C=C OMIGHNLMNHATMP-UHFFFAOYSA-N 0.000 claims description 2
- 241000186063 Arthrobacter Species 0.000 claims description 2
- 241000186146 Brevibacterium Species 0.000 claims description 2
- 241000186216 Corynebacterium Species 0.000 claims description 2
- 239000005058 Isophorone diisocyanate Substances 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims description 2
- NIMLQBUJDJZYEJ-UHFFFAOYSA-N isophorone diisocyanate Chemical compound CC1(C)CC(N=C=O)CC(C)(CN=C=O)C1 NIMLQBUJDJZYEJ-UHFFFAOYSA-N 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920001451 polypropylene glycol Polymers 0.000 claims description 2
- 241000234435 Lilium Species 0.000 claims 2
- 239000000243 solution Substances 0.000 description 10
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 241000186226 Corynebacterium glutamicum Species 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 241000186073 Arthrobacter sp. Species 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 235000010413 sodium alginate Nutrition 0.000 description 3
- 239000000661 sodium alginate Substances 0.000 description 3
- 229940005550 sodium alginate Drugs 0.000 description 3
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 3
- 235000010234 sodium benzoate Nutrition 0.000 description 3
- 239000004299 sodium benzoate Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 235000010410 calcium alginate Nutrition 0.000 description 2
- 239000000648 calcium alginate Substances 0.000 description 2
- 229960002681 calcium alginate Drugs 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920001515 polyalkylene glycol Polymers 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KMNCBSZOIQAUFX-UHFFFAOYSA-N 2-ethoxy-1,2-diphenylethanone Chemical compound C=1C=CC=CC=1C(OCC)C(=O)C1=CC=CC=C1 KMNCBSZOIQAUFX-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 235000011037 adipic acid Nutrition 0.000 description 1
- 239000001361 adipic acid Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- TXXHDPDFNKHHGW-ZPUQHVIOSA-N trans,trans-muconic acid Chemical class OC(=O)\C=C\C=C\C(O)=O TXXHDPDFNKHHGW-ZPUQHVIOSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、ムコン酸の製造方法に関する。[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing muconic acid.
（従来の技術と発明が解決しようとする問題点）
従来、安息香酸またはカテコールを酵素化学的
に反応させてムコン酸を製造することは知られて
いる。例えば、安息香酸より、コリネバクテリウ
ム属に属する変異株を用いてムコン酸を産生する
方法（醗酵工学第55巻第２号、95−97、1977記
載）、また、アルスロバクターsp.（DSM20427）
を用いる方法［アーカイブスオブマイクロバイオ
ロジー（Arch.Microbiol.）、110、253−256記載］
等が知られている。さらに、安息香酸より著量の
ムコン酸を蓄積する方法としては、アルスロバク
ター（Arthrobacter）sp.T8626（微工研菌寄第
7952号）の変異株アルスロバクター
（Arthrobacter）sp.T8626−11（微工研菌寄第
7953号）を用いた方法が知られている（特開昭60
−166902号公報記載）。(Prior Art and Problems to be Solved by the Invention) Conventionally, it has been known to produce muconic acid by enzymatically reacting benzoic acid or catechol. For example, a method for producing muconic acid from benzoic acid using a mutant strain belonging to the genus Corynebacterium (described in Fermentation Engineering Vol. )
Method using [Arch.Microbiol., 110 , 253-256]
etc. are known. Furthermore, as a method for accumulating more muconic acid than benzoic acid, Arthrobacter sp.
7952) mutant strain of Arthrobacter sp.
7953) is known (Japanese Unexamined Patent Publication No. 1983
−166902).
しかしながら、より効率良くムコン酸を製造す
る為には、上記微生物又はその処理物を固定化し
て、連続型反応器において、反応を行なわせる必
要がある。 However, in order to produce muconic acid more efficiently, it is necessary to immobilize the above-mentioned microorganism or its treated product and conduct the reaction in a continuous reactor.
近年、生体触媒の固定化担体として、極めて多
くの物質について研究が行なわれているが、それ
ぞれ一長一短があり、また、同種の担体でも固定
化する酵素、微生物等の種類及び利用する反応系
により、その活性の発現率・活性の安定性に大き
な差異がある。 In recent years, research has been conducted on a large number of substances as immobilization carriers for biocatalysts, but each has advantages and disadvantages, and even with the same type of carrier, it depends on the types of enzymes, microorganisms, etc. to be immobilized and the reaction system used. There are large differences in the expression rate and stability of the activity.
（問題点を解決するための手段）
本発明者らは、安息香酸またはカテコールから
ムコン酸を生産する能力を有する微生物またはそ
の処理物を、その活性の発現率およびその安定性
において優れた担体に包括固定することにより、
効率よく連続的にムコン酸を製造すべく研究を重
ねた結果、固定化担体として、ポリアルキレング
リコールの両末端に光重合可能なエチレン性不飽
和基を有する化合物が結合してなる光重合性樹脂
の光重合物が最も適していることを見出し、本発
明に到達した。(Means for Solving the Problems) The present inventors have developed a microorganism capable of producing muconic acid from benzoic acid or catechol, or a processed product thereof, as a carrier excellent in the expression rate of its activity and its stability. By comprehensively fixing,
As a result of repeated research to efficiently and continuously produce muconic acid, we have developed a photopolymerizable resin in which a compound having photopolymerizable ethylenically unsaturated groups is bonded to both ends of polyalkylene glycol as an immobilization carrier. The present invention was achieved by discovering that the photopolymerized product is the most suitable.
すなわち、本発明の要旨は、安息香酸またはカ
テコールを酵素化学的に反応させてムコン酸を製
造する方法において、(イ)ポリエチレングリコール
またはポリプロピレングリコールの両末端に、イ
ソホロンジイソシアナートを介してヒドロキシエ
チルアクリレートを付加させた化合物および(ロ)安
息香酸またはカテコールよりムコン酸を生成する
能力を有するアルスロバクター（Arthrobacter）
属、ミクロバクテリウム（Microbacterium）
属、ブレビバクテリウム（Brevibacterium）属、
コリネバクテリウム（Corynebacterium）属のア
セトアシドフイルム（C.acetoacidophilum）ま
たは、リリウム（C.lilium）に属する微生物また
はその処理物を、溶媒中で混合し、これに光線を
照射して上記(イ)で表わされる化合物を重合させる
ことにより得られる上記微生物またはその処理物
の包括固定化物の存在下該酵素化学的反応を行な
うことを特徴とするムコン酸の製造方法に存す
る。 That is, the gist of the present invention is to provide a method for producing muconic acid by enzymatically reacting benzoic acid or catechol. Arthrobacter has the ability to produce muconic acid from acrylate-added compounds and (bi)benzoic acid or catechol.
Genus, Microbacterium
Genus, Brevibacterium,
Microorganisms belonging to C. acetoacidophilum of the genus Corynebacterium or C. lilium or their processed products are mixed in a solvent, and the mixture is irradiated with light to produce the above (a). The method for producing muconic acid is characterized in that the enzymatic chemical reaction is carried out in the presence of an entrapping immobilized product of the microorganism or its treated product obtained by polymerizing the compound represented by the formula.
以下、本発明を詳細に説明する。 The present invention will be explained in detail below.
本発明において、固定化担体として使用する光
重合性樹脂は、ポリアルキレングリコールの両末
端に光重合可能なエチレン性不飽和基を有する化
合物が結合してなる樹脂であり、例えばポリエチ
レングリコールまたはポリプロピレングリコール
（重合度50〜70）の両末端に、イソホロンジイソ
シアナートを介して、ヒドロキシエチルアクリレ
ートを付加させた化合物、具体的には、ENT−
3400、ENTG−3800（共に、関西ペイント(株)製、
商品名）等が挙げられる。 In the present invention, the photopolymerizable resin used as the immobilization carrier is a resin in which a compound having a photopolymerizable ethylenically unsaturated group is bonded to both ends of polyalkylene glycol, such as polyethylene glycol or polypropylene glycol. A compound in which hydroxyethyl acrylate is added to both ends of (polymerization degree 50-70) via isophorone diisocyanate, specifically, ENT-
3400, ENTG-3800 (both manufactured by Kansai Paint Co., Ltd.)
product name), etc.
安息香酸またはカテコールよりムコン酸を生産
蓄積する能力のある微生物としては、例えば、ア
ルスロバクター（Arthrobacter）sp.T8626−11
（微工研菌寄第7953号）、ミクロバクテリウム・ア
ンモニアフイルム（Microbacterium
ammoniaphilum）ATCC21645、ATCC15354、
ブレビバクテリウム・フラブム
（Brevibacterium flavum）ATCC13826、ブレ
ビバクテリウム・サツカロリテイクム
（Brevibacterium saccharolyticum）
ATCC14066、ブレビバクテリウム・デイバリカ
ツム（Brevibacterium divaricatum）
ATCC21642、ブレビバクテリウム・ラクトフア
ーメンタム（Brevibacterium lactofermentum）
ATCC13655、コリネバクテリウム・アセトアシ
ドフイルム（Corynebacterium
acetoacidophilum）ATCC21421、ATCC13870、
コリネバクテリウム・リリウム
（Corynebacterium lilium）ATCC21793などが
挙げられる。 Examples of microorganisms capable of producing and accumulating muconic acid from benzoic acid or catechol include Arthrobacter sp.T8626−11.
(Feikoken Bibori No. 7953), Microbacterium ammonia film (Microbacterium ammonia film)
ammoniaphilum) ATCC21645, ATCC15354,
Brevibacterium flavum ATCC13826, Brevibacterium saccharolyticum
ATCC14066, Brevibacterium divaricatum
ATCC21642, Brevibacterium lactofermentum
ATCC13655, Corynebacterium acetoacidophilum
acetoacidophilum) ATCC21421, ATCC13870,
Examples include Corynebacterium lilium ATCC21793.
本発明においては、上記微生物自体またはその
処理物を担体に固定化させる。即ち、菌体を含む
培養液そのまま、若しくは洗浄菌体、または超音
波、トルエン若しくはリゾチーム等による処理物
等が用いられる。 In the present invention, the microorganism itself or its treated product is immobilized on a carrier. That is, a culture solution containing microbial cells as is, washed microbial cells, or a product treated with ultrasound, toluene, lysozyme, etc. is used.
これらの菌体またはその処理物を溶媒中にて前
記光重合性樹脂と混合し、光重合開始剤を加え、
好ましくは波長250〜600nｍの光を照射すること
により包括固定化物を得ることができる。その
際、光照射前に、アルギン酸ナトリウム、カラギ
ーナン等の水溶性高分子多糖類を0.1〜３％程度
加え、0.02〜1M程度の塩化カルシウム又は塩化
カリウム溶液等に適下してゲル化せしめ、その後
光照射して、洗浄すれば、取扱いやすい粒状の上
記固定化物が容易に作製できるので好ましい。 These bacterial cells or their treated products are mixed with the photopolymerizable resin in a solvent, a photopolymerization initiator is added,
Preferably, an entrapping immobilized product can be obtained by irradiating light with a wavelength of 250 to 600 nm. At that time, before irradiation with light, approximately 0.1 to 3% of water-soluble polymeric polysaccharides such as sodium alginate and carrageenan are added, and the mixture is dropped into a calcium chloride or potassium chloride solution of approximately 0.02 to 1M to form a gel. Light irradiation and washing are preferred because the above-mentioned immobilized product in the form of particles that are easy to handle can be easily produced.
菌体またはその処理物の担持量は、担体１あ
たり、乾燥重量で50ｇ程度までの範囲で任意に選
べる。 The amount of bacterial cells or their processed material supported can be arbitrarily selected within a range of up to about 50 g in dry weight per carrier.
上記光重合の際の溶媒としては、水、緩衝水溶
液、水溶性アルコール類、水溶性ケトン類及びそ
れらの混和物などの水性溶媒が好適である。 Suitable solvents for the photopolymerization are aqueous solvents such as water, aqueous buffer solutions, water-soluble alcohols, water-soluble ketones, and mixtures thereof.
かくして得られる固定化物存在下に反応させる
安息香酸濃度はナトリウム塩として、30ｇ／程
度まで、カテコール濃度は５ｇ／程度までで、
両者とも望ましくは、１ｇ／以下の範囲から選
ばれる。また、安息香酸塩以外に、種々の炭水化
物、有機酸等をさらに添加してもよく、必要に応
じて、増殖に必要な各種栄養源を用いることがで
きる。 The concentration of benzoic acid to be reacted in the presence of the immobilized product thus obtained is up to about 30 g/as a sodium salt, and the concentration of catechol is up to about 5 g/.
Both are preferably selected from a range of 1 g/or less. In addition to benzoate, various carbohydrates, organic acids, etc. may be further added, and various nutritional sources necessary for growth may be used as necessary.
反応液のPHは４−10、温度は20−40℃程度から
選ばれる。１の反応液に対して、通常、0.01〜
0.5の固定化物の存在下で反応が行なわれる。 The pH of the reaction solution is selected from 4-10, and the temperature is selected from about 20-40°C. Usually 0.01 to 1 reaction solution
The reaction is carried out in the presence of 0.5 immobilized product.
上記固定化物は回分反応でも使用可能である
が、望ましくは、これを、三相流動層、懸濁気泡
塔、懸濁撹拌槽等に投入して、連続的に反応させ
る。この際反応液中へ酸素を含むガスを吹き込む
必要がある（安息香酸１モルから、１モルのムコ
ン酸を生成するには、２モルの酸素が必要とされ
る）。 Although the above-mentioned immobilized product can be used in a batch reaction, it is preferably placed in a three-phase fluidized bed, suspension bubble column, suspension stirring tank, etc., and reacted continuously. At this time, it is necessary to blow a gas containing oxygen into the reaction solution (2 moles of oxygen are required to produce 1 mole of muconic acid from 1 mole of benzoic acid).
反応液からのムコン酸の採取・精製に際して
は、一般に有機化合物の採取・精製に用いられて
いる方法を採用することができる。 When collecting and purifying muconic acid from the reaction solution, methods generally used for collecting and purifying organic compounds can be employed.
得られたムコン酸は、水添してアジピン酸とす
ることができ、また、１，４−ジカルボン酸誘導
体等の原料として、さらには機能性樹脂原料とし
て有用である。 The obtained muconic acid can be hydrogenated to give adipic acid, and is also useful as a raw material for 1,4-dicarboxylic acid derivatives and the like, and further as a raw material for functional resins.
（発明の効果）
本発明の方法によれば、安息香酸またはカテコ
ールよりムコン酸を高効率で安定的に、しかも連
続的に製造することができる。(Effects of the Invention) According to the method of the present invention, muconic acid can be produced stably and continuously with high efficiency from benzoic acid or catechol.
（実施例）
以下、実施例により、本発明を更に詳細に説明
するが、本発明はその要旨を超えない限り以下の
実施例に限定されるものではない。(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is exceeded.
実施例１および比較例１
１の水に、ペプトン30ｇ、イーストエキス15
ｇ、NaCl10ｇを溶解し、PH7.0に調整した培地を
作製した。この50mlを500mlの三角フラスコに分
注し、120℃で20分間殺菌した。この培地に普通
ブイヨン寒天培地で30℃にて24時間生育させたア
ルスロバクターsp.T8626−11（微工研菌寄第7953
号）の２白金耳を接種し、30℃回転振盪機で17時
間培養を行つた後、安息香酸ナトリウムを５ｇ／
となるように添加して、さらに２時間培養し
た。Example 1 and Comparative Example 1 30g of peptone and 15g of yeast extract to the water from 1.
A medium was prepared by dissolving 10 g of NaCl and adjusting the pH to 7.0. This 50ml was dispensed into a 500ml Erlenmeyer flask and sterilized at 120°C for 20 minutes. On this medium, Arthrobacter sp.
After inoculating 2 platinum loops of No. 2 and culturing in a rotary shaker at 30°C for 17 hours, 5 g of sodium benzoate was added to the plate.
and cultured for an additional 2 hours.
この培養液約13mlから集菌し、0.1Mトリス塩
酸緩衝液（PH8.0）に懸濁し、３mlとした。この
懸濁液に光重合性樹脂ENTG−3800（関西ペイン
ト(株)製、商品名）7.6ｇ、３％アルギン酸ナトリ
ウム溶液1.5ｇ、ベンゾインエチルエーテル0.06
mgを加え、混合する。この混合液を0.1M塩化カ
ルシウム中へ滴下後、ゲル化した粒子に300−
400nｍの近紫外線を10分間照射した。 Bacteria were collected from about 13 ml of this culture solution and suspended in 0.1M Tris-HCl buffer (PH8.0) to make 3 ml. This suspension contains 7.6 g of photopolymerizable resin ENTG-3800 (manufactured by Kansai Paint Co., Ltd., trade name), 1.5 g of 3% sodium alginate solution, and 0.06 g of benzoin ethyl ether.
Add mg and mix. After dropping this mixture into 0.1M calcium chloride, the gelled particles were
Near ultraviolet rays of 400 nm were irradiated for 10 minutes.
このようにして作成した固定化菌体を、安息香
酸ナトリウムを２ｇ／、果糖３ｇ／、硫酸第
１鉄および硫酸マグネシウム各0.2ｍＭを含む
0.1Mトリス塩酸緩衝液（PH8.0）50mlに加えて、
500mlの三角フラスコで２時間、30℃下で振盪・
反応させた。 The thus prepared immobilized bacterial cells were mixed with sodium benzoate 2g/, fructose 3g/, ferrous sulfate and magnesium sulfate 0.2mM each.
In addition to 50ml of 0.1M Tris-HCl buffer (PH8.0),
Shake at 30℃ for 2 hours in a 500ml Erlenmeyer flask.
Made it react.
さらに、この固定化菌体を前記の増殖培地に入
れて、20〜30時間、30℃で振盪した後、上記の反
応を繰り返した。その結果、増殖後のムコン酸の
生産速度は、0.4〜0.5ｇ／／hrで安定であつ
た。 Further, the immobilized bacterial cells were placed in the above-mentioned growth medium and shaken at 30°C for 20 to 30 hours, and then the above reaction was repeated. As a result, the production rate of muconic acid after proliferation was stable at 0.4 to 0.5 g/hr.
これに対し、同様の方法で、２％のアルギン酸
ナトリウムのみで、固定化した場合は、40時間目
以降、活性が低下した（図−１参照）。 On the other hand, when immobilization was performed using only 2% sodium alginate using the same method, the activity decreased after 40 hours (see Figure 1).
実施例２および比較例２
実施例１と同様の方法で調製した固体化菌体を
三相流動層型反応器（容積1400ml）に入れて、同
様の反応液を用いて反応させた。安息香酸ナトリ
ウム濃度３〜５ｇ／、希釈率0.10〜0.15hr-1、
空気流量1.5〜2.0VVM、担体充填率0.23、平均粒
子半径0.95mmである。反応器容積あたりのムコン
酸生産性は、約110時間にわたつて、約0.3ｇ／
／hrであつた。一方、アルギン酸カルシウムゲ
ルを用いた固定化菌体反応を、ほぼ同様の条件下
で実施した場合、ムコン酸の生産性は、約70時間
にわたつて、約0.2ｇ／／hrであり、生産性、
安定性ともに劣つていた。Example 2 and Comparative Example 2 Solidified bacterial cells prepared in the same manner as in Example 1 were placed in a three-phase fluidized bed reactor (volume 1400 ml) and reacted using the same reaction solution. Sodium benzoate concentration 3-5 g/, dilution rate 0.10-0.15 hr -1 ,
The air flow rate is 1.5-2.0VVM, the carrier filling factor is 0.23, and the average particle radius is 0.95mm. Muconic acid productivity per reactor volume is approximately 0.3 g/g over approximately 110 hours.
It was /hr. On the other hand, when the immobilized bacterial cell reaction using calcium alginate gel was carried out under almost the same conditions, the productivity of muconic acid was about 0.2 g//hr over about 70 hours. ,
Both stability was poor.
図１は、光硬化性樹脂（ENTG−3800）及び、
アルギン酸カルシウムゲルに固定化したアルスロ
バクターsp.のムコン酸生産速度と培養時間との
関係を示す図である。
Figure 1 shows a photocurable resin (ENTG-3800) and
FIG. 3 is a diagram showing the relationship between muconic acid production rate and culture time of Arthrobacter sp. immobilized on calcium alginate gel.
Claims (1)
応させてムコン酸を製造する方法において、(イ)ポ
リエチレングリコールまたはポリプロピレングリ
コールの両末端に、イソホロンジイソシアナート
を介してヒドロキシエチルアクリレートを付加さ
せた化合物および(ロ)安息香酸またはカテコールよ
りムコン酸を生成する能力を有するアルスロバク
ター（Arthrobacter）属、ミクロバクテリウム
（Microbacterium）属、ブレビバクテリウム
（Brevibacterium）属、コリネバクテリウム
（Corynebacterium）属のアセトアシドフイルム
（C.acetoacidophilum）または、リリウム（C.
lilium）に属する微生物またはその処理物を、溶
媒中で混合し、これに光線を照射して上記(イ)で表
わされる化合物を重合させることにより得られる
上記微生物またはその処理物の包括固定化物の存
在下該酵素化学的反応を行なうことを特徴とする
ムコン酸の製造方法。1. In a method for producing muconic acid by enzymatically reacting benzoic acid or catechol, (a) a compound in which hydroxyethyl acrylate is added to both ends of polyethylene glycol or polypropylene glycol via isophorone diisocyanate, and (b) Acetoacids of the genus Arthrobacter, Microbacterium, Brevibacterium, and Corynebacterium, which have the ability to produce muconic acid from benzoic acid or catechol. Film (C. acetoacidophilum) or Lilium (C.
The entrapping immobilization of the above microorganisms or their processed products obtained by mixing the microorganisms belonging to the genus lilium or their processed products in a solvent and irradiating the mixture with light to polymerize the compound represented by (a) above. A method for producing muconic acid, which comprises carrying out the enzymatic chemical reaction in the presence of muconic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62028065A JPS63196296A (en) | 1987-02-12 | 1987-02-12 | Production of muconic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62028065A JPS63196296A (en) | 1987-02-12 | 1987-02-12 | Production of muconic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63196296A JPS63196296A (en) | 1988-08-15 |
| JPH0412708B2 true JPH0412708B2 (en) | 1992-03-05 |
Family
ID=12238365
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62028065A Granted JPS63196296A (en) | 1987-02-12 | 1987-02-12 | Production of muconic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63196296A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5215887A (en) * | 1975-07-23 | 1977-02-05 | Japan Atom Energy Res Inst | Process for immobilizing enzymes and microbial cells |
| JPS5247981A (en) * | 1975-10-08 | 1977-04-16 | Japan Atom Energy Res Inst | Method of immobilizing yeast or microbial cells |
| JPS61128893A (en) * | 1984-11-26 | 1986-06-16 | Agency Of Ind Science & Technol | Production of muconic acid |
-
1987
- 1987-02-12 JP JP62028065A patent/JPS63196296A/en active Granted
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5215887A (en) * | 1975-07-23 | 1977-02-05 | Japan Atom Energy Res Inst | Process for immobilizing enzymes and microbial cells |
| JPS5247981A (en) * | 1975-10-08 | 1977-04-16 | Japan Atom Energy Res Inst | Method of immobilizing yeast or microbial cells |
| JPS61128893A (en) * | 1984-11-26 | 1986-06-16 | Agency Of Ind Science & Technol | Production of muconic acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63196296A (en) | 1988-08-15 |
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