JPH02207783A - Culture of phodococcus bacteria and production of 2-ketobutyric acid using same - Google Patents
Culture of phodococcus bacteria and production of 2-ketobutyric acid using sameInfo
- Publication number
- JPH02207783A JPH02207783A JP2566089A JP2566089A JPH02207783A JP H02207783 A JPH02207783 A JP H02207783A JP 2566089 A JP2566089 A JP 2566089A JP 2566089 A JP2566089 A JP 2566089A JP H02207783 A JPH02207783 A JP H02207783A
- Authority
- JP
- Japan
- Prior art keywords
- butanediol
- ketobutyric acid
- culture
- propanediol
- bacteria
- 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 26
- 241000894006 Bacteria Species 0.000 title abstract description 6
- 238000004519 manufacturing process Methods 0.000 title description 4
- 244000005700 microbiome Species 0.000 claims abstract description 12
- BMRWNKZVCUKKSR-UHFFFAOYSA-N butane-1,2-diol Chemical compound CCC(O)CO BMRWNKZVCUKKSR-UHFFFAOYSA-N 0.000 claims abstract description 11
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 239000003125 aqueous solvent Substances 0.000 claims description 2
- 229940083957 1,2-butanediol Drugs 0.000 claims 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 abstract description 10
- 239000000047 product Substances 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 238000006911 enzymatic reaction Methods 0.000 abstract description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 abstract description 4
- 239000006228 supernatant Substances 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 abstract description 3
- 235000013772 propylene glycol Nutrition 0.000 abstract description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 2
- 235000019270 ammonium chloride Nutrition 0.000 abstract description 2
- 239000000872 buffer Substances 0.000 abstract description 2
- 239000007810 chemical reaction solvent Substances 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract 2
- 241000158504 Rhodococcus hoagii Species 0.000 abstract 1
- 241000187562 Rhodococcus sp. Species 0.000 abstract 1
- 238000013019 agitation Methods 0.000 abstract 1
- 239000001506 calcium phosphate Substances 0.000 abstract 1
- 229910000389 calcium phosphate Inorganic materials 0.000 abstract 1
- 235000011010 calcium phosphates Nutrition 0.000 abstract 1
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- NHJPVZLSLOHJDM-UHFFFAOYSA-N azane;butanedioic acid Chemical compound [NH4+].[NH4+].[O-]C(=O)CCC([O-])=O NHJPVZLSLOHJDM-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 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 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
- TYEYBOSBBBHJIV-UHFFFAOYSA-M 2-oxobutanoate Chemical compound CCC(=O)C([O-])=O TYEYBOSBBBHJIV-UHFFFAOYSA-M 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 229930182844 L-isoleucine Natural products 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical compound CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 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
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
［発明の目的Ｊ
（産業上の利用分野）
本発明は、ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓｌ属
細菌の培養方法及び該微細菌を用いた２−ケト酪酸の製
造方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Objective of the Invention J (Industrial Application Field) The present invention relates to a method for cultivating a bacterium belonging to the genus Rhodococcus and a method for producing 2-ketobutyric acid using the microbacterium.
本発明の方法によれば高収率で安価に２−ケト酪酸を製
造することができる。According to the method of the present invention, 2-ketobutyric acid can be produced in high yield and at low cost.
２−ケト酪酸は必須アミノ酸の１つであるＬ−インロイ
シンの製造原料として最も期待される前駆物質である。2-ketobutyric acid is the most promising precursor as a raw material for producing L-inleucine, which is one of the essential amino acids.
（従来の技術及び発明が解決しようとする課題）２−ケ
ト酪酸はＬ−インロイシンの原料として最も有望な前駆
物質であるが、化学合成法による製造ではプロセスが複
雑なため安価に製造することができず、Ｌ−イソロイシ
ンの原料としての工業的利用は困難な状況であった。(Prior art and problems to be solved by the invention) 2-ketobutyric acid is the most promising precursor as a raw material for L-inleucine, but it is difficult to produce it cheaply due to the complicated process of chemical synthesis. Therefore, it has been difficult to use L-isoleucine industrially as a raw material.
本発明者らはロドコッカス［Ｒｈｏｄｏｃｏｃｃｕｓｌ
属に属する微生物を培養するに際し、培地中に，２−プ
ロパンジオール及び／又は，２−ブタンジオールを添加
して培養した培養物又は該培養物から回収した菌体もし
くはその処理物の存在下、，２−ブタンジオールを水性
溶媒中で酵素反応させると２−ケト酪酸が頭重生成する
という全く新規な知見を見出し、本発明を完成するに至
った。The present inventors have discovered that Rhodococcus [Rhodococcus]
When culturing microorganisms belonging to the genus, in the presence of a culture cultured by adding 2-propanediol and/or 2-butanediol to the medium, or bacterial cells recovered from the culture or a processed product thereof, , 2-butanediol is subjected to an enzymatic reaction in an aqueous solvent to produce a head of 2-ketobutyric acid, which is a completely new finding, and the present invention has been completed.
［発明の構成］
（課題を解決するための手段及び作用）本発明は、ロド
コッカス（Ｒｈｏｄｏｃｏｃｃｕｓ） Ｍに属し、，２
−ブタンジオールから２−ケト酪酸を生成する能力を有
する微生物を培養するに際し、培地中に１゜２−プロパ
ンジオール及び／又は，２−ブタンジオールを添加する
ことを特徴とするロドコッカス［Ｒｈｏｄｏｃｏｃｃｕ
ｓｌ属に属する微生物の培養方法であり、更に、上記培
養物又は該培養物から回収した菌体もしくはその処理物
の存在下、，２−ブタンジオールから２−ケト酪酸を製
造する方法をも提供するものである。[Structure of the invention] (Means and effects for solving the problem) The present invention belongs to Rhodococcus M., 2
- When culturing a microorganism capable of producing 2-ketobutyric acid from butanediol, 1.2-propanediol and/or 2-butanediol is added to the medium.
A method for cultivating a microorganism belonging to the genus sl, and further provides a method for producing 2-ketobutyric acid from 2-butanediol in the presence of the above culture, bacterial cells recovered from the culture, or a processed product thereof. It is something to do.
本発明に用いる微生物としては、ロドコッカス（Ｒｈｏ
ｄｏｃｏｃｃｕｓｌ属に属し、，２−ブタンジオールか
ら２−ケト酪酸を生成する能力を有するものであれば特
に制限はなく、例えば、ロドコッカス・エキュイ（Ｒｈ
ｏｄｏｃｏｃｃｕｓ ｅｐｕｉ）ＩＦＯ３７３０が挙げ
られる。The microorganisms used in the present invention include Rhodococcus
There is no particular restriction as long as it belongs to the genus dococcus and has the ability to produce 2-ketobutyric acid from 2-butanediol. For example, Rhodococcus ecui (Rh
odococcus epui) IFO3730.
，２−プロパンジオール及び／又は，２−ブタンジオー
ルの培地への添加量は、培地に対して、通常０．１〜ｌ
Ｏ重量％、好ましくは０．２〜４重量％である。，２−
プロパンジオール又は，２−ブタンジオールの培地への
添加時期は特に制限されず、培養開始前であってもよく
、また培養途中であってもよい、更に、−括して添加し
てもよく、培養途中に逐次添加してもよい。, 2-propanediol and/or 2-butanediol added to the medium is usually 0.1 to 1 to the medium.
O weight %, preferably 0.2 to 4 weight %. ,2-
The timing of adding propanediol or 2-butanediol to the medium is not particularly limited, and may be before the start of culture, or during culture, or may be added all at once. It may be added sequentially during the culture.
上記の微生物を培養するための培地としては、炭素源と
してエタノール、グリセロール、酢酸又はその塩、コハ
ク酸又はその塩等を、窒素源として塩化アンモニウム、
硫酸アンモニウム、硝酸アンモニウムのような無機アン
モニウム塩、ペプトン、肉エキス、コーンステイープリ
カー、カザミノ酸のような有機窒素源等を、無機物とし
てリン酸カリウム、硫酸マグネシウム等を、また必要に
応じて各種ビタミン等の栄養素を含有した培地が好適に
使用される。なお、培地に添加する１、２−プロパンジ
オール又は，２−ブタンジオールは、２−ケト酪酸生成
酵素の生成促進物質として作用する以外に、微生物の生
育のための炭素源としても作用するので、培養開始前に
，２−プロパンジオール又は１，２−ブタンジオールを
培地中に添加しておく場合は、炭素源として前記のよう
なエタノール、グリセロール等を使用することは必ずし
も必要ではない。The medium for culturing the above microorganisms includes ethanol, glycerol, acetic acid or its salts, succinic acid or its salts as a carbon source, ammonium chloride,
Inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, peptone, meat extract, cornstarch liquor, organic nitrogen sources such as casamino acids, potassium phosphate, magnesium sulfate, etc. as inorganic substances, and various vitamins as necessary. A medium containing nutrients is preferably used. In addition, 1,2-propanediol or 2-butanediol added to the medium not only acts as a production promoter of 2-ketobutyrate-producing enzyme, but also acts as a carbon source for the growth of microorganisms. When 2-propanediol or 1,2-butanediol is added to the medium before the start of culture, it is not necessarily necessary to use ethanol, glycerol, etc. as described above as a carbon source.
培養は通気攪拌、振盪等の好気的条件下で行い、培養温
度は通常２０〜４０℃、好ましくは２５〜３５℃である
。培養途中のｐＨは通常５〜ｌＯ１好ましくは６〜８付
近であり、培養中のｐＨの調整には酸、アルカリを添加
して行うことができる。The culture is carried out under aerobic conditions such as aeration and shaking, and the culture temperature is usually 20 to 40°C, preferably 25 to 35°C. The pH during the cultivation is usually around 5 to 1O1, preferably around 6 to 8, and the pH during the cultivation can be adjusted by adding acid or alkali.
なお、培養期間は通常１〜７日間、好ましくは２〜５日
間である。In addition, the culture period is usually 1 to 7 days, preferably 2 to 5 days.
培養後、得られた培養物、該培養物から濾過又は遠心分
離により集めた菌体、該菌体の超音波等による破砕物又
は菌体もしくはその破砕物の固定化等の処理物を酵素剤
として使用する。After culturing, the obtained culture, bacterial cells collected from the culture by filtration or centrifugation, crushed products of the bacterial cells by ultrasonic waves, etc., or processed products such as immobilization of bacterial cells or their crushed materials are treated with an enzyme agent. Use as.
菌体又は菌体破砕物の固定化手段は、特に制限されるも
のではなく、例えばポリアクリルアミド、アルギン酸、
に−カラギーナン等による包括法等が好適に用いられる
。The means for immobilizing bacterial cells or crushed bacterial cells is not particularly limited, and examples include polyacrylamide, alginic acid,
An inclusive method using carrageenan or the like is preferably used.
上述の本発明に用いる各微生物は、各酵素活性を向上さ
せた変異、遺伝子組み換え、細胞融合などの手法１こよ
り得られた微生物であってもよい。Each of the microorganisms used in the present invention described above may be a microorganism obtained by one method such as mutation, genetic recombination, or cell fusion that improves each enzyme activity.
この酵素反応系には、少なくとも，２−ブタンジオール
が含まれていればよく、その濃度は例えば反応の開始時
に通常０．０１〜２０重量％、好ましくは０．０５〜ｌ
Ｏ重量％程度である。This enzyme reaction system only needs to contain at least 2-butanediol, and its concentration is usually 0.01 to 20% by weight, preferably 0.05 to 1, at the start of the reaction.
It is about 0% by weight.
，２−ブタンジオールを原料として２−ケト酪酸を生成
する微生物の培養物又は該培養物から回収した菌体もし
くはその処理物の使用量は特に限定されるものではない
が、通常０．１〜５０重量％、好ましくは１〜３０重量
％程度である。, 2-butanediol as a raw material to produce 2-ketobutyric acid, or the amount of microbial cells recovered from the culture or the processed product used is not particularly limited, but is usually 0.1 to 2. It is about 50% by weight, preferably about 1 to 30% by weight.
この反応は、ｉ）Ｈが通常５〜１０、好ましくは６〜９
で行われ１反応温度は通常的１０〜６０℃、好ましくは
約２０〜４５℃である０反応は通常約１０〜７２時間行
う。In this reaction, i) H is usually 5 to 10, preferably 6 to 9
The reaction temperature is usually 10 to 60°C, preferably about 20 to 45°C.The reaction is usually carried out for about 10 to 72 hours.
酵素反応に用いられる反応溶媒としては、水又はリン酸
もしくはトリス塩酸等の緩衝液が好ましい。The reaction solvent used in the enzyme reaction is preferably water or a buffer such as phosphoric acid or Tris-HCl.
（発明の実施例）
以下、実施例により本発明を更に詳細に説明するが、こ
れらの実施例は本発明の範囲を何ら制限するものではな
い。(Examples of the Invention) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.
以下の実施例において、２−ケト酪酸の定性は、ペーパ
ークロマトグラフのＲｆ値により、定量は高速液体クロ
マトグラフィー（島津ＬＣ−５Ａ）により行った。また
、％と表したのは重量％を意味する。In the following examples, 2-ketobutyric acid was qualitatively determined by the Rf value of a paper chromatograph, and quantitatively determined by high performance liquid chromatography (Shimadzu LC-5A). Furthermore, the expression % means weight %.
反応終了液からの２−ケト酪酸の分離・精製は、通常、
エーテル抽出法、イオン交換樹脂処理法等により行うこ
とができる。Separation and purification of 2-ketobutyric acid from the reaction completed solution is usually carried out by
This can be carried out by ether extraction method, ion exchange resin treatment method, etc.
実施例１
下記培地組成の培地１００−を５０〇−容三角フラスコ
に分注して１２０℃、１５分間滅菌処理したものに、ロ
ドコッカス・エキュイ（Ｒｈｏｄｏｃｏｃｃｕｓ ｅｐ
ｕｉｌ Ｉ Ｆ Ｏ３７３０を一白金耳量接種し、３０
℃にて２日間振盪培養（前培養とする）後、上記と同じ
培地組成Ａの培地１００〇−を５４容三角フラスコに分
注して１２０℃、１５分間滅菌処理したものに上記前培
養物２０ｄを接種したものを更に３０℃にて２日間振盪
培養した。Example 1 A 100-ml medium having the following medium composition was dispensed into a 500-ml Erlenmeyer flask and sterilized at 120°C for 15 minutes.
Inoculate one platinum loopful of uil I F O3730, 30
After shaking culture at ℃ for 2 days (referred to as pre-culture), 1000ml of medium with the same medium composition A as above was dispensed into a 54-volume Erlenmeyer flask and sterilized at 120 ℃ for 15 minutes. 20d was further cultured with shaking at 30°C for 2 days.
培養終了液１０１００Ｏ＋を遠心分離ＴＩ０，００Ｏｒ
ｐｍ、１０分、４℃）し菌体を集め、該集菌体を１７１
５Ｍリン酸緩衝液（ｐＨ７，０）５０−に懸濁後、再び
遠心分１１１ （１０，００Ｏｒｐｍ、 ｌ ０分、４
℃）して得た菌体を２−ケト酪酸生成酵素源とした。Centrifuge the culture finished solution 10,100O+ TI0,00Or
pm, 10 minutes, 4℃), collect the bacteria, and incubate the collected bacteria at 171
After suspending in 5M phosphate buffer (pH 7,0), centrifugation was performed again (10,00 rpm, 10 minutes, 4
℃) was used as a source of 2-ketobutyric acid producing enzyme.
反応液（１，２−ブタンジオール ２％、１７１５Ｍリ
ン酸緩衝液、、ｐＨ７，０）１００−に上記で調製した
２−ケト酪酸生成酵素源５ｇ（湿菌体）を加え、３０℃
にて２４時間反応を行い、該反応物から遠心分離（１０
，ＯＯＯｒｐｍ、 １０分、４℃）にて除菌した上清液
を回収し、該上清液中に生成した２−ケト酪酸量を定量
した。また、上清液５０−を塩酸酸性（ｐＨ２，０）に
調整後、該溶液にエーテル２００ｄを添加して抽出を行
った。該エーテル相を分離後、恒温水槽（５０℃）にて
エーテルを蒸発除去し２−ケト酪酸の粗精製物を得た６
以上の結果を第１表に示した。Add 5 g of the 2-ketobutyric acid-producing enzyme source (wet bacterial cells) prepared above to the reaction solution (1,2-butanediol 2%, 1715M phosphate buffer, pH 7,0) 100-100, and heat at 30°C.
The reaction was carried out for 24 hours, and the reaction product was centrifuged (10
The supernatant liquid was sterilized at 4° C., OOO rpm, 10 minutes, and 4° C.), and the amount of 2-ketobutyric acid produced in the supernatant liquid was quantified. Further, after adjusting the supernatant liquid 50- to acidic with hydrochloric acid (pH 2,0), 200 d of ether was added to the solution for extraction. After separating the ether phase, the ether was removed by evaporation in a constant temperature water bath (50°C) to obtain a crude product of 2-ketobutyric acid 6
The above results are shown in Table 1.
１皿■戒
基本培地 酵母エキス ０，０１％ポリペプト
ン ０．５ ％
（ＮＨ４１ＪＰＯ−０、６５％
ＫＪ（、ＰＯ，０，３５％
ＭｇＳＯ４・７Ｈ，００，０４％
ＦｅＳＯ４４Ｈ＊０ ４５ ｐｐｍＭｎＳ
Ｏ４・４Ｈｉ０ ５ ｐｐ１＋１ＺｎＳ
Ｏａ４Ｈｘ０ ３０ ｐｐｍＣｕＳＯ４’
５Ｈ＊０ ２．５ ｐｐｍＮａＣｌ
５０ ｐｐｍＣａＣＯ５０，５％
蒸留水 １０００ １ｄＩ
（ｐＨ７，０）
Ａ：基本培地にエタノール１容量％添加、ｐＨ７，０
Ｂ：基本培地にエタノールｌ容量％と，２−ブタンジオ
ール１％添加、ｐｉ−１’７．０Ｃ：基本培地にグリセ
ロール１％添加、ｐＨ７、Ｏ
Ｄ二基本培地にグリセロール１％と，２−ブタンジオー
ル１％添加、ｐＨ７，０
Ｅ：基本培地にコハク酸アンモニウム１％添加、ｐＨ７
，０
Ｆ：基本培地にコハク酸アンモニウム１％と，２−ブタ
ンジオール１％添加、ＰＨ
７，０
Ｇ；基本培地にエタノールｌ容量％と，２−プロパンジ
オール１％添加、ｐ）（’７．ＯＨ：基本培地にグリセ
ロール１％と，２−プロパンジオール１％添加、ｐＨ７
，０
に基本培地に，２−ブタンジオール１％添加、ｐＨ７，
０
Ｊ：基本培地に，２−プロパンジオール１％添加、ｐＨ
７，０
に；基本培地に，２−プロパンジオール１％と，２−ブ
タンジオール１％添加、ｐＨ
７，０
上記第１表から、培地に，２−ブタンジオール又は，２
−プロパンジオールを添加することにより２−ケト酪酸
生成量が顕著に増大することがわかる。1 dish ■Kai basic medium Yeast extract 0.01% Polypeptone 0.5% (NH41JPO-0, 65% KJ (, PO, 0.35% MgSO4・7H, 00.04% FeSO44H*0 45 ppmMnS
O4・4Hi0 5 pp1+1ZnS
Oa4Hx0 30 ppmCuSO4'
5H*0 2.5 ppmNaCl
50 ppm CaCO50.5% Distilled water 1000 1 dI (pH 7.0) A: Addition of 1 volume % of ethanol to the basic medium, pH 7.0 B: Addition of 1 volume % of ethanol and 1% of 2-butanediol to the basic medium, pi-1 '7.0C: 1% glycerol added to basic medium, pH 7, OD 1% glycerol and 1% 2-butanediol added to basic medium, pH 7.0 E: 1% ammonium succinate added to basic medium, pH 7
, 0 F: Addition of 1% ammonium succinate and 1% 2-butanediol to the basic medium, PH 7,0 G; Addition of 1% ethanol by volume and 1% 2-propanediol to the basic medium, p) ('7 .OH: 1% glycerol and 1% 2-propanediol added to basic medium, pH 7
, 0 to the basic medium, 1% 2-butanediol added, pH 7,
0 J: 1% 2-propanediol added to basic medium, pH
7.0; 1% 2-propanediol and 1% 2-butanediol were added to the basic medium, pH 7.0 From Table 1 above, 2-butanediol or 2-butanediol was added to the medium.
It can be seen that the amount of 2-ketobutyric acid produced increases significantly by adding -propanediol.
［発明の効果］
本発明の方法によれば、安価な，２−ブタンジオールか
ら好効率で安価に２−ケト酪酸を製造することができる
。[Effects of the Invention] According to the method of the present invention, 2-ketobutyric acid can be produced efficiently and inexpensively from inexpensive 2-butanediol.
一：回収されず1: Not collected
Claims (2)
し、１，２−ブタンジオールから２−ケト酪酸を生成す
る能力を有する微生物を培養するに際し、培地中に１，
２−プロパンジオール及び／又は１，２−ブタンジオー
ルを添加することを特徴とするロドコッカス属に属し、
１，２−ブタンジオールから２−ケト酪酸を生成する能
力を有する微生物の培養方法。(1) When culturing a microorganism that belongs to the genus Rhodococcus and has the ability to produce 2-ketobutyric acid from 1,2-butanediol, 1,
Belongs to the genus Rhodococcus, characterized by the addition of 2-propanediol and/or 1,2-butanediol,
A method for culturing a microorganism capable of producing 2-ketobutyric acid from 1,2-butanediol.
し、１，２−ブタンジオールから２−ケト酪酸を生成す
る能力を有する微生物の培養物又は該培養物から回収し
た菌体もしくはその処理物の存在下、１，２−ブタンジ
オールを水性溶媒中で反応させて２−ケト酪酸を生成さ
せた後、該反応液から２−ケト酪酸を採取することを特
徴とする２−ケト酪酸の製造方法。(2) In the presence of a culture of a microorganism belonging to the genus Rhodococcus and having the ability to produce 2-ketobutyric acid from 1,2-butanediol, or bacterial cells recovered from the culture, or a processed product thereof, 1 , 2-butanediol in an aqueous solvent to produce 2-ketobutyric acid, and then collecting 2-ketobutyric acid from the reaction solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2566089A JP2670130B2 (en) | 1989-02-06 | 1989-02-06 | Method for culturing Rhodococcus bacteria and method for producing 2-ketobutyric acid using the microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2566089A JP2670130B2 (en) | 1989-02-06 | 1989-02-06 | Method for culturing Rhodococcus bacteria and method for producing 2-ketobutyric acid using the microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02207783A true JPH02207783A (en) | 1990-08-17 |
| JP2670130B2 JP2670130B2 (en) | 1997-10-29 |
Family
ID=12171963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2566089A Expired - Lifetime JP2670130B2 (en) | 1989-02-06 | 1989-02-06 | Method for culturing Rhodococcus bacteria and method for producing 2-ketobutyric acid using the microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2670130B2 (en) |
-
1989
- 1989-02-06 JP JP2566089A patent/JP2670130B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2670130B2 (en) | 1997-10-29 |
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