JPH03183476A - Production of 2-ketobutyric acid - Google Patents
Production of 2-ketobutyric acidInfo
- Publication number
- JPH03183476A JPH03183476A JP32329189A JP32329189A JPH03183476A JP H03183476 A JPH03183476 A JP H03183476A JP 32329189 A JP32329189 A JP 32329189A JP 32329189 A JP32329189 A JP 32329189A JP H03183476 A JPH03183476 A JP H03183476A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- ketobutyric acid
- ketobutyric
- dihydroxybutyric
- microorganism
- 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
- TYEYBOSBBBHJIV-UHFFFAOYSA-N 2-oxobutanoic acid Chemical compound CCC(=O)C(O)=O TYEYBOSBBBHJIV-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 22
- LOUGYXZSURQALL-UHFFFAOYSA-N 2,3-dihydroxybutanoic acid Chemical compound CC(O)C(O)C(O)=O LOUGYXZSURQALL-UHFFFAOYSA-N 0.000 claims abstract description 12
- 241000589516 Pseudomonas Species 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 239000000758 substrate Substances 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 20
- 238000000034 method Methods 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 241000589776 Pseudomonas putida Species 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- DLVZBSZXZDGKQY-UHFFFAOYSA-N 2,2-dihydroxybutanoic acid Chemical compound CCC(O)(O)C(O)=O DLVZBSZXZDGKQY-UHFFFAOYSA-N 0.000 description 3
- STAWYILKMSIACI-UHFFFAOYSA-N 3,3-dihydroxybutanoic acid Chemical compound CC(O)(O)CC(O)=O STAWYILKMSIACI-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical group O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- LDHQCZJRKDOVOX-NSCUHMNNSA-N crotonic acid Chemical compound C\C=C\C(O)=O LDHQCZJRKDOVOX-NSCUHMNNSA-N 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- LDHQCZJRKDOVOX-UHFFFAOYSA-N trans-crotonic acid Natural products CC=CC(O)=O LDHQCZJRKDOVOX-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012285 osmium tetroxide Substances 0.000 description 2
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- XUJJIYQHKOCLOJ-ARJAWSKDSA-N (z)-2-methylsulfonyloxybut-2-enoic acid Chemical compound C\C=C(C(O)=O)/OS(C)(=O)=O XUJJIYQHKOCLOJ-ARJAWSKDSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 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 1
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- RENMDAKOXSCIGH-UHFFFAOYSA-N Chloroacetonitrile Chemical compound ClCC#N RENMDAKOXSCIGH-UHFFFAOYSA-N 0.000 description 1
- 108700016168 Dihydroxy-acid dehydratases Proteins 0.000 description 1
- 101100001672 Emericella variicolor andG gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108090001042 Hydro-Lyases Proteins 0.000 description 1
- 102000004867 Hydro-Lyases Human genes 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 241000108056 Monas Species 0.000 description 1
- LFTLOKWAGJYHHR-UHFFFAOYSA-N N-methylmorpholine N-oxide Chemical compound CN1(=O)CCOCC1 LFTLOKWAGJYHHR-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229940124277 aminobutyric acid Drugs 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- WYACBZDAHNBPPB-UHFFFAOYSA-N diethyl oxalate Chemical compound CCOC(=O)C(=O)OCC WYACBZDAHNBPPB-UHFFFAOYSA-N 0.000 description 1
- -1 diethyl propionate Chemical compound 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 238000007040 multi-step synthesis reaction Methods 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (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）
本発明は新規な微生物と該微生物による２−ケト酪酸の
製造法に関する。２−ケト酪酸は例えばインロイシン発
酵の原料あるいは光学活性なヒドロキシ酪酸、光学活性
なアミノ酪酸、抗生物質であるバウロマイシンＡなどの
原料となる。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel microorganism and a method for producing 2-ketobutyric acid using the microorganism. 2-ketobutyric acid is, for example, a raw material for inleucine fermentation, or a raw material for optically active hydroxybutyric acid, optically active aminobutyric acid, and the antibiotic bauromycin A.
（従来の技術）
従来、２−ケト酪酸を工業的に合成する方法としては、
シュウ酸ジエチルとプロピオン酸ジエチルを脱水縮合さ
せた後、濃硫酸で加水分解する方法が知られている。こ
のほか、アセトアルデヒドとシアノメチルクロライドを
出発物質として多段階で得る方法や、２−メタンスルホ
ニルオキシクロトン酸を加水分解する方法、エトキサリ
ルプロビオン酸エチルエステルを加水分解する方法等が
公知である。(Prior art) Conventionally, methods for industrially synthesizing 2-ketobutyric acid include:
A method is known in which diethyl oxalate and diethyl propionate are subjected to dehydration condensation and then hydrolyzed with concentrated sulfuric acid. In addition, there are known methods such as a multi-step method using acetaldehyde and cyanomethyl chloride as starting materials, a method of hydrolyzing 2-methanesulfonyloxycrotonic acid, and a method of hydrolyzing ethoxalylprobionic acid ethyl ester.
また、上記の化学合成法とは別に、微生物を用いる方法
については、アエロバクタ−・アエロバクタ（Ａｅｒｏ
ｂａｃｔｅｒ ａｅｒｏｇｅｎｅｓ）、ミクロコツカス
・リソデイクトリカス（Ｍｌｃｒｏｃｏｃｃｕｓ Ｉｙ
ｓｏｄｅｌｋｔｒｌｃｕｓ）、トルロプシス・ニーティ
リス（丁ｏｒｕｌｏｐｓ１ｓ ｕｔｌ目Ｓ）、ｆｆ１．
ッシェリシア・コリ（Ｅｓｃｈｅｌｌｃｈｌａ ｃｏｌ
ｌ）、サツカロマイセス・セレビシェ（Ｓａｃｃｈａｒ
ｏｍｙｃｅｓ ｃｅｒｅｖｉｓｌａｅ）などが産出する
ジヒドロキシ酸デヒドラターゼ（Ｄｌｈｙｄｒｏｘｙａ
ｃｌｄ ｄｅｂｙｄｒａｔａｓｅ（４，２，１，９）
）が、２．３−ジヒドロキシ酪酸を基質とし、これを２
−ケト酪酸に変換する能力を持つことなどが知られてい
る（Ｗｌｘｏｍ、 Ｒ，Ｌ、 ｅｔ ａｌ、、 Ｂｉｏ
ｃｈｉｍ。In addition to the above-mentioned chemical synthesis method, there is also a method using microorganisms such as Aerobacter
bacter aerogenes), Micrococcus lithodeictricus (Mlcrococcus Iy)
sodelktrlcus), Torulopsis nitilis (Torullops 1s utl. S), ff1.
Eschellchla col
l), Saccharomyces cerevisiae (Sacchar
dihydroxy acid dehydratase (Dlhydroxyacid dehydratase) produced by
cld debydratase (4, 2, 1, 9)
) uses 2,3-dihydroxybutyric acid as a substrate and converts it into 2
- It is known that it has the ability to convert into ketobutyric acid (Wlxom, R,L, et al., Bio
chim.
Ｂ（ｏｐｈｙｓ、 Ａｃｔａ、ｉｌ、４３３ （１９８
１） ）。B(ophys, Acta, il, 433 (198
1) ).
（発明が解決しようとする問題点）
これら従来の方法は、化学合成法では、多段階の合成を
必要としたり、出発原料が高価であったりするため工業
的に有利な方法ではなく、また、微生物を用いる方法で
は、生成収率が低いためにやはり工業生産には適さない
。そこで、安価な合成法の確立が求められていた。本発
明は、従来法よりも安価に２−ケト酪酸の製造を可能な
らしめるものである。(Problems to be Solved by the Invention) These conventional chemical synthesis methods are not industrially advantageous because they require multi-step synthesis and the starting materials are expensive. Methods using microorganisms have low production yields and are therefore not suitable for industrial production. Therefore, there was a need to establish an inexpensive synthesis method. The present invention makes it possible to produce 2-ketobutyric acid at a lower cost than conventional methods.
Ｃ問題点を解決するための手段）
本発明者らは、２−ケト酪酸製造の原料として安価なり
ロトン酸から容易にトレオ体またはエリスロ体として有
機合成される２、３−ジヒドロキシ酪酸に着目した。そ
して、２，３−ジヒドロキシ酪酸を効率よく２−ケト酪
酸に変換する酵素系をもつ微生物を得るために、基質と
してトレオ体を用いて広く天然界から検索した結果、シ
ュードモナス・プチダに属する微生物が、トレオー２゜
３−ジヒドロキシ酪酸を効率よく２−ケト酪酸に変換す
る酵素系をもつことを見いだし、発明を完成した。Means for Solving Problem C) The present inventors focused on 2,3-dihydroxybutyric acid, which is inexpensive and easily organically synthesized as a threo or erythro form from rotonic acid as a raw material for producing 2-ketobutyric acid. . In order to obtain a microorganism with an enzyme system that efficiently converts 2,3-dihydroxybutyric acid into 2-ketobutyric acid, we conducted a wide search in nature using threo bodies as a substrate, and found that a microorganism belonging to the Pseudomonas putida family was found. They discovered that they have an enzyme system that efficiently converts threo-2°3-dihydroxybutyric acid into 2-ketobutyric acid, and completed the invention.
すなわち、本発明はシュードモナス属に属し、２．３−
ジヒドロキシ酪酸を２−ケト酪酸に変換する能力を有す
る微生物であり、また当該微生物あるいはその処理物の
存在下に、２，３−ジヒドロキシ酪酸を２−ケト酪酸に
変換して２−ケト酪酸を蓄積せしめ、これを採取するこ
とを特徴とする２−ケト酪酸の製造法である。That is, the present invention belongs to the genus Pseudomonas, 2.3-
It is a microorganism that has the ability to convert dihydroxybutyric acid to 2-ketobutyric acid, and in the presence of the microorganism or its processed product, it converts 2,3-dihydroxybutyric acid to 2-ketobutyric acid and accumulates 2-ketobutyric acid. This is a method for producing 2-ketobutyric acid, which is characterized by collecting the 2-ketobutyric acid.
従来、２，３−ジヒドロキシ酪酸を２−ケト酪酸に変換
する酵素系をシュードモナス属細菌が産生ずることは公
知ではなかった。したがって、２゜３−ジヒドロキシ酪
酸を２−ケト酪酸に変換する酵素系をもつ本願の微生物
は、新規なものである。It was not previously known that bacteria of the genus Pseudomonas produce an enzyme system that converts 2,3-dihydroxybutyric acid to 2-ketobutyric acid. Therefore, the microorganism of the present application having an enzyme system that converts 2°3-dihydroxybutyric acid to 2-ketobutyric acid is novel.
本発明に使用される酵素系は、シュードモナス属に属す
る細菌が有する２、３−ジヒドロキシ酪酸を２−ケト酪
酸に変換する作用を利用するものである。The enzyme system used in the present invention utilizes the ability of bacteria belonging to the genus Pseudomonas to convert 2,3-dihydroxybutyric acid to 2-ketobutyric acid.
本発明者らは、トレオー２，３−ジヒドロキシ酪酸を効
率よく２−ケト酪酸に変換する酵素系をもつ微生物を天
然界から検索した結果、５Ｄ３Ａ株（微工研菌寄第目１
２Ｇ号）を単離した。５Ｄ３Ａ株は、トレオー２，３−
ジヒドロキシ酪酸を２−ケト酪率よく蓄積するためには
有利であり、５Ｄ３Ａ株（微工研菌寄第１１１２８号）
は、本発明に用いる微生物として好ましいものである。As a result of searching for microorganisms in the natural world that have an enzyme system that efficiently converts threo-2,3-dihydroxybutyric acid into 2-ketobutyric acid, the present inventors found that the 5D3A strain (Feikoken Bacteria
2G) was isolated. The 5D3A strain is threo2,3-
It is advantageous to accumulate dihydroxybutyric acid at a high 2-ketobutyric rate, and the 5D3A strain (Feikoken Bibori No. 11128)
are preferable as microorganisms used in the present invention.
５Ｄ３Ａ株（微工研菌寄第１１１２Ｇ号）の菌学的性質
を以下に示す。The mycological properties of the 5D3A strain (Feikoken Bibori No. 1112G) are shown below.
以上の菌学的諸性質を基準として、Ｂｅｒｇｅｙ’５）
Ｉａｎｕａｌ ｏｆ Ｄｅｔｅｒｖｌｎａｔｌｖｅ Ｂ
ａｃｔｅｒｉｏｌｏｇｙ 第８版（１９７４）の分類
基準により検索すると、５Ｄ３Ａ株（微工研菌寄第１１
１２８号）は、シュードモナス・プチダ（Ｐｓｅｕｄｏ
ｍｏｎａｓ ｐｕｔｉｄａ）であると同定された。Based on the above mycological properties, Bergey'5)
Annual of Detervlnatlve B
When searching according to the classification criteria of acteriology 8th edition (1974), strain 5D3A (Feikoken Bacterial Collection No. 11) was found.
No. 128) is Pseudomonas putida (Pseudomonas putida).
Monas putida).
本発明は、シュードモナス属に属する微生物を使用して
、２−ケト酪酸を製造する方法に関するものである。The present invention relates to a method for producing 2-ketobutyric acid using a microorganism belonging to the genus Pseudomonas.
本発明において使用するシュードモナス属に属し、２，
３−ジヒドロキシ酪酸を２−ケト酪酸に変換する能力を
有する微生物としては、上記シュードモナス・プチダ（
Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ）５Ｄ３Ａ株（
シュードモナス・プチダ 丁Ｅ３４８７とも呼ぶ）（微
工研菌寄第１１１２１１ｉ号）が例示されるが、該菌株
に限定されない。該微生物の処理物とは、該微生物の培
養物から精製された酵素ならびに該酵素の固定化物など
を意味する。Belongs to the genus Pseudomonas used in the present invention, 2,
Examples of microorganisms that have the ability to convert 3-dihydroxybutyric acid to 2-ketobutyric acid include the above-mentioned Pseudomonas putida (
Pseudomonas putida) 5D3A strain (
An example is Pseudomonas putida (also referred to as Ding E3487) (Feikoken Bibori No. 111211i), but it is not limited to this strain. The processed product of the microorganism refers to an enzyme purified from a culture of the microorganism, an immobilized product of the enzyme, and the like.
本発明において原料となるトレオ（またはエリスロ）−
２，３−ジヒドロキシ酪酸は、一般にクロトン酸から合
成される。合成法については、４酸化オスミウム触媒の
存在下に、クロトン酸をＮ−メチルモルホリン−Ｎ−オ
キサイドによって酸化する方法（Ｆｒａｎｋ Ｂ、 Ａ
ｒｍｓｔｒｏｎｇ、 ｅｔ ａｌ、、 Ｊ。Threo (or erythro), which is the raw material in the present invention
2,3-dihydroxybutyric acid is generally synthesized from crotonic acid. Regarding the synthesis method, a method in which crotonic acid is oxidized with N-methylmorpholine-N-oxide in the presence of an osmium tetroxide catalyst (Frank B, A
rmstrong, et al., J.
Ｃｈｅｗ、 Ｓｏｃ、 Ｐｅｒｋｉｎ Ｔｒａｎ
ｓ、 Ｉ、 １９８５． ８９１（１９８５）
）また、４酸化オスミウムと過酸化水素の存在下にクロ
トン酸から合成する方法（Ｆ、Ｗ、 Ｂａｃｈｅｌｏｒ
ａｎｄＧ、Ａ、旧ａｎａ、 ｃａｎａｄｉａｎ Ｊ、
Ｃｈｅｗ、、 柱、 ４０８９（１９８９））などが公
知である。Chew, Soc, Perkin Tran
s, I, 1985. 891 (1985)
) Also, a method of synthesis from crotonic acid in the presence of osmium tetroxide and hydrogen peroxide (F, W, Bachelor
andG, A, former ANA, canadian J,
Chew, Pillar, 4089 (1989)) are well known.
本発明の微生物を培養するための培地組成としては、炭
素源・窒素源・無機物及び必要に応じて少量の微量栄養
素を含むものであれば、合成培地または天然培地のいず
れも使用可能である。培地に使用する炭素源としては、
グリセリン、グルコース、可溶性でんぷん等、資化され
るものならばいずれも可能である。トレオ（またはエリ
スロ）＝２，３−ジヒドロキシ酪酸は、それ自身は炭素
源にならないが、この反応系に関与する酵素系を誘導す
る効果があり、培地中に添加することが好ましい。また
、窒素源としては、アンモニウム塩、硝酸塩等が用いら
れる。たとえば、硝酸アンモニウムまたは硫酸アンモニ
ウムである。無機イオンとしては、リン酸イオン、マグ
ネシウムイオン、カリウムイオン、その他が適宜用いら
れる。さらに、有機栄養素として、酵母エキス、ペプト
ン、肉エキス、コ、−ンスティープリカーなどが適宜用
いられる。As the medium composition for culturing the microorganism of the present invention, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, inorganic substances, and, if necessary, a small amount of micronutrients. The carbon source used in the culture medium is
Anything that can be assimilated, such as glycerin, glucose, and soluble starch, can be used. Although threo (or erythro)=2,3-dihydroxybutyric acid does not itself serve as a carbon source, it has the effect of inducing the enzyme system involved in this reaction system, and is therefore preferably added to the medium. Further, as the nitrogen source, ammonium salts, nitrates, etc. are used. For example, ammonium nitrate or ammonium sulfate. As the inorganic ions, phosphate ions, magnesium ions, potassium ions, and others are used as appropriate. Further, as organic nutrients, yeast extract, peptone, meat extract, corn, corn steep liquor, etc. are used as appropriate.
上記培地にて、本発明の微生物をｐＨ６〜８（好ましく
は７．２）、温度１７〜２８℃（好ましくは２５℃）に
おいて１２〜２４時間培養する。調製された微生物の培
養物は、トレオ（またはエリスロ）−２，３−ジヒドロ
キシ酪酸を２−ケト酪酸に変換する酵素系をもつもので
ある。このような培養物はそのまま酵素源として反応に
使用してもよく、また、培養物を遠心分離などで分離し
て得られる生菌株、その乾燥菌体、あるいは、分離した
菌体を超音波処理・自己消化・磨砕などの方法により処
理して得られる菌体処理物、さらにはこれらの菌体より
の抽出物ならびに該抽出物より得られる酵素の粗製物ま
たは精製物として使用してもよい。もちろん、これらの
固定化菌体または固定化酵素としても使用できる。The microorganism of the present invention is cultured in the above medium at a pH of 6 to 8 (preferably 7.2) and a temperature of 17 to 28°C (preferably 25°C) for 12 to 24 hours. The microbial culture prepared has an enzyme system that converts threo (or erythro)-2,3-dihydroxybutyric acid to 2-ketobutyric acid. Such a culture may be used as it is in the reaction as an enzyme source, or a live bacterial strain obtained by separating the culture by centrifugation, dried bacterial cells, or separated bacterial cells may be treated with ultrasonication.・It may be used as a treated bacterial cell product obtained by processing methods such as autolysis or grinding, or as an extract from these bacterial cells, as well as a crude or purified enzyme obtained from the extract. . Of course, these can also be used as immobilized bacterial cells or immobilized enzymes.
本発明方法ではこのようにして調製した菌体、菌体処理
物、固定化菌体などを触媒として用い、トレオ（または
エリスロ）−２，３−ジヒドロキシ酪酸を反応液中で変
換せしめて、２−ケト酪酸を生成蓄積せしめる。In the method of the present invention, the thus prepared bacterial cells, treated bacterial cells, immobilized bacterial cells, etc. are used as catalysts to convert threo (or erythro)-2,3-dihydroxybutyric acid in the reaction solution. -Produces and accumulates ketobutyric acid.
反応液には、２−ケト酪酸の生成率を高めるために、基
質であるトレオ（またはエリスロ） −２，３−ジヒド
ロキシ酪酸の他に、２ａｉカチオンの添加が好ましく、
例えば反応液中に１ｍＭの塩化コバルトを共存させるこ
とにより、著しく生成効率を高めることができる。基質
の使用量には制限が無いが、好ましくは１００＋Ｍであ
る。酵素反応は、通常ｐＨ８〜１０（好ましくは９．０
）、温度は１０〜４０℃（好ましくは２５℃）において
行う。In order to increase the production rate of 2-ketobutyric acid, it is preferable to add a 2ai cation to the reaction solution in addition to the substrate threo (or erythro)-2,3-dihydroxybutyric acid.
For example, by coexisting 1 mM cobalt chloride in the reaction solution, the production efficiency can be significantly increased. There is no limit to the amount of substrate used, but it is preferably 100+M. The enzyme reaction is usually carried out at a pH of 8 to 10 (preferably 9.0).
), at a temperature of 10 to 40°C (preferably 25°C).
反応時間は、目的に応じ適宜設定されるものであるが、
通常１〜２０時間である。The reaction time is set appropriately depending on the purpose, but
Usually 1 to 20 hours.
得られた反応液からの２−ケト酪酸の採取方法は、例え
ば、反応液を遠心分離した上清をクロマトグラフィーに
より分画する等の方法に従って行うことができる。2-ketobutyric acid can be collected from the obtained reaction solution, for example, by centrifuging the reaction solution and fractionating the supernatant by chromatography.
（実施例）
以下、実施例を挙げて説明するが、これらは例示であっ
て本発明を限定するものではない。(Example) Examples will be described below, but these are illustrative and do not limit the present invention.
なお、トレオー２，３−ジヒドロキシ酪酸の定量は高速
液体クロマトグラフ法で、２−ケト酪酸の定量は、２．
４−ジニトロフェニルヒドラジンを用いて分光光度法に
より行った。The determination of threo-2,3-dihydroxybutyric acid was performed using high performance liquid chromatography, and the determination of 2-ketobutyric acid was performed using 2.
Performed spectrophotometrically using 4-dinitrophenylhydrazine.
実施例 １
グリセリン０．５％、トレオー２，３−ジヒドロキシ酪
酸０．３％１酵母エキス０．１５％、硫酸アンモニウム
０．１％、リン酸−カリウム０．１５％、リン酸二カリ
ウム０．１５％、食塩０．１％、硫酸マグネシウム七水
塩０，０２％ほか微量要素を含む培地（ｐＨ７，２）を
肩付きフラスコに５０−人れ、１２０℃で２０分間殺菌
した。これにシュードモナス、プチダ５Ｄ３Ａ株（微工
研菌寄第１１１２１１ｉ号）を接種し、２５℃で１２時
間、振とう培養した。この培養液より菌体を遠心分離に
より採取した後ｔｌｏｏｍＭ）レオ−２，３−ジヒドロ
キシ酪酸を含む１００ｍＭ）リス−塩酸緩衝液（ｐＨ９
，０）で、菌体濃度が４０■／ＩＬｌになるように懸濁
した。また、懸濁の際には同時に塩化コバルトを濃度１
ｍＭになるよう加えた（全量１０ｗＬり。この反応液を
、振とうしながら２５℃、０〜２４時間保持し、２−ケ
ト酪酸生成量を測定した。結果を第１表に示す。Example 1 Glycerin 0.5%, threo-2,3-dihydroxybutyric acid 0.3% 1 yeast extract 0.15%, ammonium sulfate 0.1%, potassium phosphate 0.15%, dipotassium phosphate 0.15 %, salt 0.1%, magnesium sulfate heptahydrate 0.02%, and other trace elements (pH 7.2) was poured into a shoulder flask for 50 minutes and sterilized at 120°C for 20 minutes. This was inoculated with Pseudomonas putida strain 5D3A (Feikoken Bibori No. 111211i), and cultured with shaking at 25°C for 12 hours. After collecting the bacterial cells from this culture solution by centrifugation, a solution of 100 mM) Lis-HCl buffer (pH 9) containing rheo-2,3-dihydroxybutyric acid was added.
, 0), and the cells were suspended at a bacterial cell concentration of 40 μ/IL. Also, during suspension, cobalt chloride at a concentration of 1
The reaction solution was kept at 25° C. for 0 to 24 hours while shaking, and the amount of 2-ketobutyric acid produced was measured. The results are shown in Table 1.
第 １ 表
また、菌体濃度が１０，２０．８０ｍｇ／−の場合でも
同様の操作を行った。その結果を、菌体濃度４０■／ｊ
の場合を含めて、第１図に示す。Table 1 Also, the same operation was performed when the bacterial cell concentration was 10 and 20.80 mg/-. The result is a bacterial cell concentration of 40■/j
This is shown in Figure 1, including the case of .
第１図は、遠心分離により採取した菌体を、１００＋ｌ
Ｉ）レオ−２，３−ジヒドロキシ酪酸を含む１００■関
トリス−塩酸緩衝液（ｐＨ９，０）で、菌体濃度がそれ
ぞれ１０１２０％ ４０１８０ｗ／ｌＺにまなるように
懸濁し、また、懸濁の際に同時に塩化コバルトを濃度１
ｍＭになるよう加えた反応液（全量１０−）を、振とう
しながら２５℃、０〜２４時間保持した場合の２−ケト
酪酸生成量を示したものである。Figure 1 shows 100+l of bacterial cells collected by centrifugation.
I) Suspend the bacteria in 100 μL Kanto Tris-HCl buffer (pH 9,0) containing rheo-2,3-dihydroxybutyric acid so that the cell concentration becomes 10,120% and 40,180 w/lZ, respectively. At the same time, cobalt chloride was added at a concentration of 1.
The figure shows the amount of 2-ketobutyric acid produced when a reaction solution (total amount 10-) added to make an mM concentration was maintained at 25° C. for 0 to 24 hours while shaking.
図中の記号〇−〇は菌体濃度１０■／−の場合を、以下
Δ−Δは２ Ｏｎ ／ ｄ 、ローロは４０■／−１・
−・は８０ｍｇ／−の場合をそれぞれ表す。The symbols 〇-〇 in the figure are for the case where the bacterial cell concentration is 10■/-, below Δ-Δ is 2 On / d, and Rollo is 40■/-1.
-・represents the case of 80 mg/-, respectively.
実施例 ２
グリセリン０．５％、酵母エキス０．１５％、硫酸アン
モニウム０．１％、りん酸−カリウム０．１５％、りん
酸二カリウム０．１５％、食塩０．１％、硫酸マグネシ
ウム七水塩０．０２％ばか微量要素を含む培地（ｐＨ７
，２）に、さらにＯ〜２．Ｏｄの範囲でトレオー２，３
−ジヒドロキシ酪酸を添加して、肩つきフラスコに５０
−入れ、１２０℃で２０分間殺菌した。それぞれにシュ
ードモナス・プチダ５Ｄ３Ａ株（微工研菌寄第１１１２
１１ｉ号）を接種し、２５℃で１２時間振とう培養のの
ち、菌体を遠心分離により集めた。Example 2 Glycerin 0.5%, yeast extract 0.15%, ammonium sulfate 0.1%, potassium phosphate 0.15%, dipotassium phosphate 0.15%, salt 0.1%, magnesium sulfate heptahydrate Medium containing 0.02% salt (pH 7)
, 2), and further O~2. Treau 2,3 in the range of Od
- Add dihydroxybutyric acid to a shouldered flask for 50 min.
- and sterilized at 120°C for 20 minutes. Pseudomonas putida 5D3A strain (Feikoken Bacterial Serial No. 1112)
No. 11i) was inoculated and cultured with shaking at 25°C for 12 hours, and then the bacterial cells were collected by centrifugation.
集めた菌体を、１００ｍＭ）レオ−２，３−ジヒドロキ
シ酪酸を含む１００＋＋＋Ｍ）リス−塩酸緩衝液（ｐＨ
９，０）で、菌体濃度が２０■／−になるようにそれぞ
れ懸濁した。また、懸濁の際には同時に塩化コバルトを
濃度１ｍ旧こなるよう加えた（全量１ＯＴＬり。これら
の反応を、振とうしながら２５℃、２４時間保持し、２
−ケト酪酸生成量を測定した。結果を第２表に示す。The collected bacterial cells were diluted with 100+++M) Lis-HCl buffer (pH
9,0), and the cells were suspended at a bacterial cell concentration of 20/-. At the same time, cobalt chloride was added at a concentration of 1 m2 during suspension (total volume was 1 OTL). These reactions were maintained at 25°C for 24 hours with shaking, and
-The amount of ketobutyric acid produced was measured. The results are shown in Table 2.
第
表
（発明の効果）
本発明は、新規な微生物ならびに該微生物による２−ケ
ト酪酸の製造法を提供するものであり、実施例において
示されたごとく、効率よく２−ケト酪酸の生成が可能で
あることが明らかである。Table 1 (Effects of the Invention) The present invention provides a novel microorganism and a method for producing 2-ketobutyric acid using the microorganism, and as shown in the Examples, 2-ketobutyric acid can be produced efficiently. It is clear that
第１図は、２−ケト酪酸生成量を示す。 FIG. 1 shows the amount of 2-ketobutyric acid produced.
Claims (2)
酪酸を２−ケト酪酸に変換する能力を有する微生物。(1) A microorganism that belongs to the genus Pseudomonas and has the ability to convert 2,3-dihydroxybutyric acid to 2-ketobutyric acid.
酪酸を２−ケト酪酸に変換する能力を有する微生物ある
いはその処理物の存在下に、２，３−ジヒドロキシ酪酸
を２−ケト酪酸に変換して２−ケト酪酸を蓄積せしめ、
これを採取することを特徴とする２−ケト酪酸の製造法
。(2) Converting 2,3-dihydroxybutyric acid to 2-ketobutyric acid in the presence of a microorganism that belongs to the genus Pseudomonas and has the ability to convert 2,3-dihydroxybutyric acid to 2-ketobutyric acid, or a processed product thereof. Accumulates 2-ketobutyric acid,
A method for producing 2-ketobutyric acid, which comprises collecting the 2-ketobutyric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32329189A JPH0822222B2 (en) | 1989-12-12 | 1989-12-12 | 2-Ketobutyric acid manufacturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32329189A JPH0822222B2 (en) | 1989-12-12 | 1989-12-12 | 2-Ketobutyric acid manufacturing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03183476A true JPH03183476A (en) | 1991-08-09 |
| JPH0822222B2 JPH0822222B2 (en) | 1996-03-06 |
Family
ID=18153146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP32329189A Expired - Lifetime JPH0822222B2 (en) | 1989-12-12 | 1989-12-12 | 2-Ketobutyric acid manufacturing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0822222B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461544A (en) * | 2016-01-28 | 2016-04-06 | 天津科技大学 | Method for extracting 2-ketobutyric acid from enzymatic conversion solution |
-
1989
- 1989-12-12 JP JP32329189A patent/JPH0822222B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461544A (en) * | 2016-01-28 | 2016-04-06 | 天津科技大学 | Method for extracting 2-ketobutyric acid from enzymatic conversion solution |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0822222B2 (en) | 1996-03-06 |
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