JPH0146112B2 - - Google Patents
Info
- Publication number
- JPH0146112B2 JPH0146112B2 JP11559280A JP11559280A JPH0146112B2 JP H0146112 B2 JPH0146112 B2 JP H0146112B2 JP 11559280 A JP11559280 A JP 11559280A JP 11559280 A JP11559280 A JP 11559280A JP H0146112 B2 JPH0146112 B2 JP H0146112B2
- Authority
- JP
- Japan
- Prior art keywords
- hydroxyisobutyric acid
- genus
- geotrichum
- endomyces
- microorganisms
- 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
Links
- 241000159512 Geotrichum Species 0.000 claims description 22
- 241000178951 Endomyces Species 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 12
- 241000235648 Pichia Species 0.000 claims description 11
- 244000005700 microbiome Species 0.000 claims description 11
- DBXBTMSZEOQQDU-UHFFFAOYSA-N 3-hydroxyisobutyric acid Chemical compound OCC(C)C(O)=O DBXBTMSZEOQQDU-UHFFFAOYSA-N 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 241000235395 Mucor Species 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 7
- 241001467578 Microbacterium Species 0.000 claims description 7
- 241000192041 Micrococcus Species 0.000 claims description 7
- 241000222068 Sporobolomyces <Sporidiobolaceae> Species 0.000 claims description 7
- 241000223230 Trichosporon Species 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 244000168141 Geotrichum candidum Species 0.000 claims description 6
- 235000017388 Geotrichum candidum Nutrition 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 241001509401 Gordonia rubripertincta Species 0.000 claims description 5
- 244000286779 Hansenula anomala Species 0.000 claims description 5
- 235000014683 Hansenula anomala Nutrition 0.000 claims description 5
- 241000221523 Rhodotorula toruloides Species 0.000 claims description 5
- 241000698291 Rugosa Species 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 241000352744 Magnusiomyces tetrasperma Species 0.000 claims description 3
- 241000187654 Nocardia Species 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 241000223252 Rhodotorula Species 0.000 claims description 2
- 150000001298 alcohols Chemical class 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 235000014633 carbohydrates Nutrition 0.000 claims description 2
- 150000007524 organic acids Chemical class 0.000 claims description 2
- 235000005985 organic acids Nutrition 0.000 claims description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims 2
- 239000006285 cell suspension Substances 0.000 claims 1
- 239000012531 culture fluid Substances 0.000 claims 1
- 229930195733 hydrocarbon Natural products 0.000 claims 1
- 150000002430 hydrocarbons Chemical class 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 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 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 241000235062 Pichia membranifaciens Species 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000222173 Candida parapsilosis Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000005273 aeration Methods 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
- 229940055022 candida parapsilosis Drugs 0.000 description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000005696 Diammonium phosphate Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 2
- 235000019838 diammonium phosphate Nutrition 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- -1 mannitrate Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229960001763 zinc sulfate Drugs 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241001634940 Apiotrichum loubieri Species 0.000 description 1
- 241000222175 Diutina rugosa Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000150860 Hyphopichia burtonii Species 0.000 description 1
- 241000191953 Kocuria varians Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241001236817 Paecilomyces <Clavicipitaceae> Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000228390 Sporobolomyces johnsonii Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241001634945 Trichosporon aquatile Species 0.000 description 1
- 239000000061 acid fraction Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- TXHIDIHEXDFONW-UHFFFAOYSA-N benzene;propan-2-one Chemical compound CC(C)=O.C1=CC=CC=C1 TXHIDIHEXDFONW-UHFFFAOYSA-N 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
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007806 chemical reaction intermediate Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 229930003231 vitamin Natural products 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
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、光学活性炭素を有する種々の天然物
または医薬品などの生理活性物質を合成する際に
有用な反応中間体の１つである光学活性なＤ(-)
−β−ヒドロキシイソ酪酸の微生物を利用した工
業的に有利な製造方法に関するものである。
従来、光学活性なＤ(-)またはＬ(+)−β−ヒ
ドロキシイソ酪酸類を製造する方法としては、光
学活性アミン類などのような分割剤を用いてDL
（±）−β−ヒドロキシイソ酪酸を光学分割する方
法が知られている。しかしながらこの公知の方法
は、使用される分割剤が高価であるばかりか、そ
の操作が極めて煩雑であるので工業的に有利な方
法でない。本発明方法によれば簡単な操作によつ
て光学的に高純度なＤ(-)−β−ヒドロキシイソ
酪酸が高収率で得られる。
即ち本発明は、DL(±)−β−ヒドロキシイソ
酪酸を、このものを立体特異的にＬ(+)−β−ヒ
ドロキシイソ酪酸を分解する能力を有するキヤン
デイダ属、ハンゼヌラ属、ピチア属、ロードスポ
リデイウム属、スポロボロミセス属、スポリデイ
オボルス属、トリコスポロン属、エンドミセス
属、ゲオトリクム属、ムコル属、ペシロミセス
属、ミクロバクテリウム属、ミクロコツカス属、
ノカルデイア属に属する微生物に接触させること
を特徴とするＤ(-)−β−ヒドロキシイソ酪酸の
製造方法に関するものである。
本発明に使用される微生物は、キヤンデイダ
属、ハンゼヌラ属、ピチア属、ロードスポリデイ
ウム属、スポロボロミセス属、スポリデイオボル
ス属、トリコスポロン属、エンドミセス属、ゲオ
トリクム属、ムコル属、ペシロミセス属、ミクロ
バクテリウム属、ミクロコツカス属、ノカルデイ
ア属に属し、Ｌ(+)−β−ヒドロキシイソ酪酸を
分解する能力を有する微生物なら何でも良いが、
例えば、キヤンデイダ・パラプシロシス
（Candida parapsilosis）IFO1022、キヤンデイ
ダ・ルゴーザ（Candida rugosa）IFO1542、ハ
ンゼヌラ・アノマラ（Hansenula anomala）
IFO0146、ピチア・メンブラナエフアシエンス
（Pichia membranaefaciens）IAM4258、ピチ
ア・ブルトニー（Pichia burtonii）IFO0844、ロ
ードスポリデイウム・トルロイデス
（Rhodosporidium toruloides）IFO0559、スポ
ロボロミセス・サルモニコラー
（Sprobolomyces salmonicolor）IAM12249、ス
ポリデイオボルス・ジヨンソニー
（Sporidiobolus johnsonii）IFO6903、トリコス
ポロン・アキユアチル（Trichosporon
aquatile）ATCC22130、エンドミセス・オベテ
ンシス（Endomyces ovetensis）IFO1201、エン
ドミセス・ゲオトリクム（Endomyces
geotrichum）CBS178、71、エンドミセス・レー
シー（Endomyces reessii）CBS179、60、エン
ドミセス・テトラスパーマ（Endomyces
tetrasperma）CBS765、70、ゲオトリクム・ア
ミセリクム（Geotrichum amycelicum）
CBS186、38、ゲオトリクム・キヤンデイドウム
（Geotrichum Candidum）CBS187、67、ゲオト
リクム・ロウビエリ（Geotrichum loubieri）
CBS252、61、ゲオトリクム・バンリエイ
（Geotrichum vanryiae）CBS439、64、ムコ
ル・アルターナンス（Mucor alternans）
HUT1115、ペシロミセス・バリオテイ
（Paecilomyces varioti）HUT4018、ミクロバク
テリウム・ルテウス（Microbacterium luteus）
IFO3064、ミクロコツカス・バリアンス
（Micrococcus varians）IFO3765、ノカルデイ
ア・コラリナ（Nocardia corallina）IFO3338等
が使用される。この培養には、通常これらの菌が
資化しうる栄養源なら何でも使用しうるが、例え
ば炭素源としてグルコース、シユクロース、マン
ニツト、デンプン等の炭水化物、エタノールを始
めとするアルコール、パラフイン・オレフイン類
の炭化水素、酢酸・ソイ酪酸等の有機酸、大豆油
等またこれらの混合物、窒素源として硫酸アンモ
ニウム・リン酸アンモニウム等、有機栄養源とし
てイーストエキス・麦芽エキス・肉エキス・ペプ
トン等、また微量金属塩、ビタミン等、通常の培
養に用いられる栄養源を適宜混合した培地を用い
ることができる。
培養の方法としては、栄養培地のPH3.0〜9.0の
範囲で好気的に20〜40℃の範囲で１〜５日間培養
する。DL(±)−β−ヒドロキシイソ酪酸からＤ
（−）−β−ヒドロキシイソ酪酸製造方法として
は、菌体の培養と並行して行なう方法として、例
えばDL(±)−β−ヒドロキシイソ酪酸を唯一の
炭素源とするか、または上記の炭素源との共存下
でPH3.0〜9.0の範囲で好気的に培養し、培養液中
よりＤ(-)−β−ヒドロキシイソ酪酸を得る方法
があり、また菌体の培養とDL(±)−β−ヒドロ
キシイソ酪酸からＤ(-)−β−ヒドロキシイソ酪
酸を得る反応とを分けて２段階で行なう方法、例
えば菌体の生産を炭素源としてDL(±)−β−ヒ
ドロキシイソ酪酸単独あるいは資化しうる上記の
炭素源、またはこれらの混合物の栄養培地でPH
3.0〜9.0の範囲で好気的に培養し、得られた培養
液にDL(±)−β−ヒドロキシイソ酪酸を添加し、
PHを3.0〜9.0に保持して好気的に反応せしめる方
法、または得られた培養液から遠心分離等で菌体
を集め菌体を適当な組成の液、例えばＭ／15リン
酸緩衝液等に懸濁し、DL(±)−β−ヒドロキシ
イソ酪酸と少量のグルコースを加え好気的にPH
3.0〜9.0の範囲で反応を行なう方法がある。この
場合の菌体は反応速度を早める為にトルエン処理
等の適当な前処理を加えたものも使用できる。
培養及び反応で得られたＤ(-)−β−ヒドロキ
シイソ酪酸の採取方法としては、通常の公知の抽
出精製方法が利用しうるが、次の如き方法も使用
しうる。例えば、得られたＤ(-)−β−ヒドロキ
シイソ酪酸含有液のPHを硫酸等で1.0付近まで下
げ、更に飽和となる様に硫酸アンモニウムを加え
る。しかる後２倍容量の酢酸エチルで３回抽出を
行なう。これを低温、減圧下にて溶剤を除き、Ｄ
（−）−β−ヒドロキシイソ酪酸含有物が油状で得
られ、更にこのものを少量のベンゼンに溶解し、
ベンゼン−アセトン混合溶剤で溶出するシリカゲ
ルクロマトグラフイーを行なう事により容易に他
の不純物と分離する事ができる。
β−ヒドロキシイソ酪酸の定量は、ジエーム
ズ・アール・シエーフアー（James Ｒ・
Schaeffer）等の方法（Biotechnology and
Bioengineering）13、208、1971）に準じて行な
つた。
次に、本発明を実施例によつて説明するが、本
発明は実施例のみに限定されるものではない。
実施例 １
グルコース２％、イーストエキス0.5％、ペプ
トン0.3％、肉エキス0.3％、Ｌ(+)−β−ヒドロ
キシイソ酪酸0.1％含有する培地（PH6.0）１
に、夫々キヤンデイダ・パラプシロシス
IFO1022、キヤンデイダ・ルゴーザIFO1542、ハ
ンゼヌラ・アノマラIFO0146、ピチア・メンブラ
ナエフアシエンスIAM4258、ピチア・ブルトニ
ーIFO0844、ロードスポリデイウム・トルロイデ
スIFO0559、スポロボロミセス・サルモニコラー
IAM12249、スポリデイオボルス・ジヨンソニー
IFO6903、トリコスポロン・アキユアチル
ATCC22130、エンドミセス・オベテンシス
IFO1201、エンドミセス・ゲオトリクムCBS178、
71、エンドミセス・レーシーCBS179、60、エン
ドミセス・テトラスパーマCBS765、70、ゲオト
リクム・アミセリクムCBS186、38、ゲオトリク
ム・キヤンデイドウムCBS187、67、ゲオトリク
ム・ロウビエリCBS252、61、ゲオトリクム・バ
ンリエイCBS439、64、ムコル・アルターナンス
HUT1115、ペシロミセス・バリオテイ
HUT4018、ミクロバクテリウム・ルテウス
IFO3064、ミクロコツカス・バリアンス
IFO3765、ノカルデイア・コラリナIFO3338を接
種し、３容ミニジヤーフアメンターで30℃、通
気1VVM、撹拌500rpmで20時間培養した。その
後、各培養液にDL(±)−β−ヒドロキシイソ酪
酸30ｇを添加し、カセイソーダでPH6.0調整して
更に48時間反応を行なつた。得られた反応液を濃
硫酸でPH1.0とし硫酸アンモニウムを加え飽和溶
液とした。次に等量の酢酸エチルで３回抽出を
し、抽出液を無水硫酸ソーダで脱水し、これを減
圧下、50℃以下で溶剤を除去し黄色油状物質を得
た。
この油状物質を重量５倍のシリカゲル（ワコー
ゲルQ50）を用いベンゼンで調製したカラムにか
けた。その後、ベンゼン：アセトン（３：１）溶
剤でβ−ヒドロキシイソ酪酸を溶出した。得られ
たβ−ヒドロキシイソ酪酸画分を集め、減圧下、
溶剤を除去しシロツプ状の物質を得た。
この様に得られたものは、ガスクロマトグラフ
イー、シリカゲル薄層クロマトグラフイー、
NMR分析により高純度なβ−ヒドロキシイソ酪
酸である事が確認された。次に、このβ−ヒドロ
キシイソ酪酸の旋光度をユニオン技研製のデジタ
ル自動旋光度計PM101にて測定した結果は、表
１に示す如くＤ(-)−β−ヒドロキシイソ酪酸で
ある事が確認された。
The present invention relates to optically active D(-), which is one of the reaction intermediates useful in the synthesis of various natural products having optically active carbon or physiologically active substances such as pharmaceuticals.
The present invention relates to an industrially advantageous production method of -β-hydroxyisobutyric acid using microorganisms. Conventionally, the method for producing optically active D(-) or L(+)-β-hydroxyisobutyric acids has been to use a resolving agent such as an optically active amine.
A method for optically resolving (±)-β-hydroxyisobutyric acid is known. However, this known method is not industrially advantageous because the resolving agent used is expensive and the operation is extremely complicated. According to the method of the present invention, optically highly pure D(-)-β-hydroxyisobutyric acid can be obtained in high yield through simple operations. That is, the present invention provides DL(±)-β-hydroxyisobutyric acid for use in plants of the genus Candeida, genus Hansenula, genus Pichia, and Rhodos, which have the ability to stereospecifically decompose L(+)-β-hydroxyisobutyric acid. Polydeium, Sporobolomyces, Sporideiobolus, Trichosporon, Endomyces, Geotrichum, Mucor, Pecilomyces, Microbacterium, Micrococcus,
The present invention relates to a method for producing D(-)-β-hydroxyisobutyric acid, which comprises bringing it into contact with a microorganism belonging to the genus Nocardia. The microorganisms used in the present invention include the genus Candeida, Hansenula, Pichia, Rhodosporidium, Sporobolomyces, Sporideiobolus, Trichosporon, Endomyces, Geotrichum, Mucor, and Pecilomyces. Any microorganism that belongs to the genus Microbacterium, Micrococcus, or Nocardia and has the ability to decompose L(+)-β-hydroxyisobutyric acid may be used.
For example, Candida parapsilosis IFO1022, Candida rugosa IFO1542, Hansenula anomala
IFO0146, Pichia membranaefaciens IAM4258, Pichia burtonii IFO0844, Rhodosporidium toruloides IFO0559, Sprobolomyces salmonicolor IAM12249, Sporideio Sporidiobolus johnsonii IFO6903, Trichosporon
aquatile) ATCC22130, Endomyces ovetensis IFO1201, Endomyces geotrichum
geotrichum) CBS178, 71, Endomyces reessii CBS179, 60, Endomyces tetrasperm (Endomyces
tetrasperma) CBS765, 70, Geotrichum amycelicum
CBS186, 38, Geotrichum Candidum CBS187, 67, Geotrichum loubieri
CBS252, 61, Geotrichum vanryiae CBS439, 64, Mucor alternans
HUT1115, Paecilomyces varioti HUT4018, Microbacterium luteus
IFO3064, Micrococcus varians IFO3765, Nocardia corallina IFO3338, etc. are used. For this culture, any nutrient source that can be assimilated by these bacteria can be used, but for example, carbohydrates such as glucose, sucrose, mannitrate, and starch, alcohols such as ethanol, and carbonized paraffins and olefins can be used as carbon sources. Hydrogen, organic acids such as acetic acid and soybutyric acid, soybean oil and mixtures thereof, nitrogen sources such as ammonium sulfate and ammonium phosphate, organic nutritional sources such as yeast extract, malt extract, meat extract, peptone, etc., trace metal salts, A medium containing an appropriate mixture of nutrients such as vitamins used in normal culture can be used. As a method of culturing, the cells are cultured aerobically at a pH of 3.0 to 9.0 in a nutrient medium at a temperature of 20 to 40°C for 1 to 5 days. DL(±)-β-hydroxyisobutyric acid to D
(−)-β-Hydroxyisobutyric acid can be produced by using DL(±)-β-hydroxyisobutyric acid as the sole carbon source, or by using the above-mentioned carbon There is a method to obtain D(-)-β-hydroxyisobutyric acid from the culture solution by culturing aerobically in the pH range of 3.0 to 9.0 in the presence of a bacterial cell culture and DL (± )-β-hydroxyisobutyric acid to D(-)-β-hydroxyisobutyric acid in two steps, for example, DL(±)-β-hydroxyisobutyric acid using bacterial cell production as a carbon source. PH in a nutrient medium of the above carbon sources alone or assimilated, or a mixture of these.
Cultivate aerobically in the range of 3.0 to 9.0, add DL(±)-β-hydroxyisobutyric acid to the resulting culture solution,
A method of maintaining the pH between 3.0 and 9.0 and reacting aerobically, or collecting the bacterial cells from the obtained culture solution by centrifugation, etc. Add DL(±)-β-hydroxyisobutyric acid and a small amount of glucose, and adjust the pH aerobically.
There is a method of conducting the reaction in the range of 3.0 to 9.0. In this case, the bacterial cells may be subjected to appropriate pretreatment such as toluene treatment in order to accelerate the reaction rate. As a method for collecting D(-)-β-hydroxyisobutyric acid obtained through the culture and reaction, conventional extraction and purification methods can be used, but the following methods can also be used. For example, the pH of the resulting D(-)-β-hydroxyisobutyric acid-containing solution is lowered to around 1.0 with sulfuric acid or the like, and ammonium sulfate is added to achieve saturation. Thereafter, extraction is carried out three times with twice the volume of ethyl acetate. The solvent was removed from this under reduced pressure at low temperature, and D
A substance containing (-)-β-hydroxyisobutyric acid was obtained in the form of an oil, which was further dissolved in a small amount of benzene,
It can be easily separated from other impurities by silica gel chromatography eluting with a benzene-acetone mixed solvent. The determination of β-hydroxyisobutyric acid was performed by James R.
Schaeffer et al.'s method (Biotechnology and
Bioengineering) 13 , 208, 1971). Next, the present invention will be explained with reference to Examples, but the present invention is not limited to the Examples. Example 1 Medium containing 2% glucose, 0.5% yeast extract, 0.3% peptone, 0.3% meat extract, and 0.1% L(+)-β-hydroxyisobutyric acid (PH6.0) 1
and Candida parapsilosis, respectively.
IFO1022, Quillandida rugosa IFO1542, Hansenula anomala IFO0146, Pichia membranaefaciens IAM4258, Pichia burtoni IFO0844, Rhodosporidium toruloides IFO0559, Sporobolomyces salmonicolara
IAM12249, Sporideiobolus jionsonii
IFO6903, Trichosporon aquiyuachil
ATCC22130, Endomyces obetensis
IFO1201, Endomyces geotrichum CBS178,
71, Endomyces lacei CBS179, 60, Endomyces tetraperma CBS765, 70, Geotrichum amycelicum CBS186, 38, Geotrichum kyandidium CBS187, 67, Geotrichum rowbieri CBS252, 61, Geotrichum vanlieri CBS439, 64, Mucor alternance
HUT1115, Pecilomyces variotei
HUT4018, Microbacterium luteus
IFO3064, Micrococcus variance
IFO3765 and Nocardia corallina IFO3338 were inoculated and cultured in a 3-volume mini-jar fermenter at 30°C, aeration at 1 VVM, and stirring at 500 rpm for 20 hours. Thereafter, 30 g of DL(±)-β-hydroxyisobutyric acid was added to each culture solution, the pH was adjusted to 6.0 with caustic soda, and the reaction was continued for an additional 48 hours. The resulting reaction solution was adjusted to pH 1.0 with concentrated sulfuric acid, and ammonium sulfate was added to make a saturated solution. Next, extraction was performed three times with equal amounts of ethyl acetate, the extract was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure at below 50°C to obtain a yellow oily substance. This oily substance was applied to a column prepared with benzene using 5 times the weight of silica gel (Wako Gel Q50). Thereafter, β-hydroxyisobutyric acid was eluted with a benzene:acetone (3:1) solvent. The obtained β-hydroxyisobutyric acid fractions were collected and separated under reduced pressure.
Removal of the solvent gave a syrupy substance. The products obtained in this way can be used for gas chromatography, silica gel thin layer chromatography,
NMR analysis confirmed that it was highly pure β-hydroxyisobutyric acid. Next, the optical rotation of this β-hydroxyisobutyric acid was measured using a digital automatic polarimeter PM101 manufactured by Union Giken, and the results confirmed that it was D(-)-β-hydroxyisobutyric acid as shown in Table 1. It was done.
【表】【table】
【表】
実施例 ２
DL(±)−β−ヒドロキシイソ酪酸2.0％、グル
コース2.0％、リン酸二アンモニウム1.3％、リン
酸一カリウム0.7％、塩化ナトリウム0.1％、硫酸
亜鉛0.006％、硫酸第一鉄0.009％、イーストエキ
ス0.3％、（PH6.5）１に、夫々キヤンデイダ・
パラプシロシスIFO1022、キヤンデイダ・ルゴー
ザIFO1542、ハンゼヌラ・アノマラIFO0146、ピ
チア・メンブラナエフアシエンスIAM4258、ピ
チア・ブルトニーIFO0844、ロードスポリデイウ
ム・トルロイデスIFO0559、スポロボロミセス・
サルモニコラーIAM12249、スポリデイオボル
ス・ジヨンソニーIFO6903、トリコスポロン・ア
キユアチルATCC22130、エンドミセス・オベテ
ンシスIFO1201、エンドミセス・ゲオトリクム
CBS178、71、エンドミセス・レーシーCBS179、
60、エンドミセス・テトラスパーマCBS765、
70、ゲオトリクム・アミセリクムCBS186、38、
ゲオトリクム・キヤンデイドウムCBS187、67、
ゲオトリクム・ロウビエリCBS252、61、ゲオト
リクム・バンリエイCBS439、64、ムコル・アル
ターナンスHUT1115、ペシロミセス・バリオテ
イHUT4018、ミクロバクテリウム・ルテウス
IFO3064、ミクロコツカス・バリアンス
IFO3765、ノカルデイア・コラリナIFO3338を接
種し、３容ミニジヤーフアメンターにて30℃、
通気1VVM、撹拌500rpmで24時間培養、その後
PHを6.0〜7.0にコントロール（カセイソーダ及び
硫酸）し、さらに48時間培養を続けた。
表２に示す如く、この培養液を実施例１と同様
に処理し、Ｄ(-)−β−ヒドロキシイソ酪酸を得
た。[Table] Example 2 DL(±)-β-hydroxyisobutyric acid 2.0%, glucose 2.0%, diammonium phosphate 1.3%, monopotassium phosphate 0.7%, sodium chloride 0.1%, zinc sulfate 0.006%, dibasic sulfate Iron 0.009%, yeast extract 0.3%, (PH6.5) 1, and Candeida, respectively.
parapsilosis IFO1022, Quillandida rugosa IFO1542, Hansenula anomala IFO0146, Pichia membranaefaciens IAM4258, Pichia burtoni IFO0844, Rhodosporidium toruloides IFO0559, Sporobolomyces
Salmonicola IAM12249, Sporideiobolus jiyonsonii IFO6903, Trichosporon achiyuachil ATCC22130, Endomyces obetensis IFO1201, Endomyces geotrichum
CBS178, 71, End Mrs. LacyCBS179,
60, Endomyces tetraperma CBS765,
70, Geotrichum amycelicum CBS186, 38,
Geotrichum kyandidoum CBS187, 67,
Geotrichum rowvieri CBS252, 61, Geotrichum banlieri CBS439, 64, Mucor alternans HUT1115, Pecilomyces variotei HUT4018, Microbacterium luteus
IFO3064, Micrococcus variance
Inoculated with IFO3765 and Nocardia corallina IFO3338, and incubated at 30℃ in a 3-volume mini-jar fermenter.
Incubate for 24 hours with aeration 1VVM and stirring 500 rpm, then
The pH was controlled at 6.0 to 7.0 (caustic soda and sulfuric acid), and culture was continued for an additional 48 hours. As shown in Table 2, this culture solution was treated in the same manner as in Example 1 to obtain D(-)-β-hydroxyisobutyric acid.
【表】【table】
【表】【table】
【表】
実施例 ３
グルコース4.0％、リン酸二アンモニウム1.3
％、リン酸−カリウム0.7％、塩化ナトリウム
0.01％、硫酸亜鉛0.006％、硫酸第一鉄0.009％、
イーストエキス0.3％、含有培地（PH6.5）１
に、夫々キヤンデイダ・パラプシロシス
IFO1022、キヤンデイダ・ルゴーザIFO1542、ハ
ンゼヌラ・アノマラIFO0146、ピチア・メンブラ
ナエフアシエンスIAM4258、ピチア・ブルトニ
ーIFO0844、ロードスポリデイウム・トルロイデ
スIFO0559、スポロボロミセス・サルモニコラー
IAM12249、スポリデイオボルス・ジヨンソニー
IFO6903、トリコスポロン・アキユアチル
ATCC22130、エンドミセス・オベテンシス
IFO1201、エンドミセス・ゲオトリクムCBS178、
71、エンドミセス・レーシーCBS179、60、エン
ドミセス・テトラスパーマCBS765、70、ゲオト
リクム・アミセリクムCBS186、38、ゲオトリク
ム・キヤンデイドウムCBS187、67、ゲオトリク
ム・ロウビエリCBS252、61、ゲオトリクム・バ
ンリエイCBS439、64、ムコル・アルターナンス
HUT1115、ペシロミセス・バリオテイ
HUT4018、ミクロバクテリウム・ルテウス
IFO3064、ミクロコツカス・バリアンス
IFO3765、ノカルデイア・コラリナIFO3338を接
種し、30℃、通気1VVM、撹拌700rpmで20時間
培養し、得られた培養液を遠心分離により菌体を
集め、更に0.9％食塩水で２回洗浄した菌体を得
た。これをＭ／15リン酸緩衝液（PH6.0）に懸濁
し、DL(±)−β−ヒドロキシイソ酪酸30ｇ、グ
ルコース２ｇ添加し、PH6.0に調整し培養と同一
条件で48時間反応させた。その後実施例１と同様
な方法で抽出精製を行ない表３の如くＤ(-)−β
−ヒドロキシイソ酪酸を得た。[Table] Example 3 Glucose 4.0%, diammonium phosphate 1.3
%, potassium phosphate 0.7%, sodium chloride
0.01%, zinc sulfate 0.006%, ferrous sulfate 0.009%,
Yeast extract 0.3%, medium containing (PH6.5) 1
and Candida parapsilosis, respectively.
IFO1022, Quillandida rugosa IFO1542, Hansenula anomala IFO0146, Pichia membranaefaciens IAM4258, Pichia burtoni IFO0844, Rhodosporidium toruloides IFO0559, Sporobolomyces salmonicolara
IAM12249, Sporideiobolus jionsonii
IFO6903, Trichosporon aquiyuachil
ATCC22130, Endomyces obetensis
IFO1201, Endomyces geotrichum CBS178,
71, Endomyces lacei CBS179, 60, Endomyces tetrasperma CBS765, 70, Geotrichum amycelicum CBS186, 38, Geotrichum kyandidium CBS187, 67, Geotrichum rowbieri CBS252, 61, Geotrichum vanlieri CBS439, 64, Mucor alternance
HUT1115, Pecilomyces variotei
HUT4018, Microbacterium luteus
IFO3064, Micrococcus variance
IFO3765 and Nocardia corallina IFO3338 were inoculated and cultured for 20 hours at 30°C, aeration 1 VVM, and stirring 700 rpm. The resulting culture solution was centrifuged to collect bacterial cells, and the bacterial cells were further washed twice with 0.9% saline. I got it. This was suspended in M/15 phosphate buffer (PH6.0), 30 g of DL(±)-β-hydroxyisobutyric acid and 2 g of glucose were added, the pH was adjusted to 6.0, and the mixture was allowed to react for 48 hours under the same conditions as culture. Ta. After that, extraction and purification was performed in the same manner as in Example 1, and D(-)-β was obtained as shown in Table 3.
-Hydroxyisobutyric acid was obtained.
【表】【table】
【表】【table】
Claims (1)
ンデイダ属、ハンゼヌラ属、ピチア属、ロードス
ポリデイウム属、スポロボロミセス属、スポリデ
イオボルス属、トリコスポロン属、エンドミセス
属、ゲオトリクム属、ムコル属、ペシロミセス
属、ミクロバクテリウム属、ミクロコツカス属、
ノカルデイア属に属し、かつＬ(+)−β−ヒドロ
キシイソ酪酸分解能を有する微生物に接触させる
ことによりＬ(+)−β−ヒドロキシイソ酪酸を分
解し、残存するＤ(-)−β−ヒドロキシイソ酪酸
を採取する事を特徴とするＤ(-)−β−ヒドロキ
シイソ酪酸の製造方法。 ２ DL(±)−β−ヒドロキシイソ酪酸を添加し
た培地中で微生物を培養することにより、DL
（±）−β−ヒドロキシイソ酪酸と微生物とを接触
させる特許請求の範囲第１項記載の製造方法。 ３ 微生物を栄養培地で培養して得た培養液を
DL(±)−β−ヒドロキシイソ酪酸に作用させる
特許請求の範囲第１項記載の製造方法。 ４ 微生物を栄養培地で培養して得た培養液から
微生物菌体を分離して菌体懸濁液を調製し、それ
をDL(±)−β−ヒドロキシイソ酪酸に作用させ
る特許請求の範囲第１項記載の製造方法。 ５ 微生物がキヤンデイダ・パラプシロシス、キ
ヤンデイダ・ルゴーザ、ハンゼヌラ・アノマラ、
ピチア・メムブラナエフアシエンス、ピチア・ブ
ルトニー、ロードスポリデイウム・トルロイデ
ス、スポロボロミセス・サルモニコラー、スポリ
デイオボルス・ジヨンソニー、トリコスポロン・
アキユアチル、エンドミセス・オベテンシス、エ
ンドミセス・ゲオトリクム、エンドミセス・レー
シー、エンドミセス・テトラスパーマ、ゲオトリ
クム・アミセリクム、ゲオトリクム・キヤンデイ
ドウム、ゲオトリクム・ロウビエリ、ゲオトリク
ム・バンリエイ、ムコル・アルターナンス、ペシ
ロミセス・バリオテイ、ミクロバクテリウム・ル
テウス、ミクロコツカス・バリアンス、ノカルデ
イア・コラリナである特許請求の範囲第１項記載
の製造方法。 ６ 微生物の炭素源としてDL(±)−β−ヒドロ
キシイソ酪酸単独または、これに資化しうる炭水
化物、アルコール、有機酸または炭化水素の１種
または２種以上を共存せしめたものを使用する特
許請求の範囲第１項〜５項記載の製造方法。[Scope of Claims] 1 DL(±)-β-hydroxyisobutyric acid can be used in the genus Candeida, genus Hansenula, genus Pichia, genus Rhodosporidium, genus Sporobolomyces, genus Sporidioborus, genus Trichosporon, and Endomyces. Genus, Geotrichum, Mucor, Pecilomyces, Microbacterium, Micrococcus,
L(+)-β-hydroxyisobutyric acid is decomposed by contacting with a microorganism that belongs to the genus Nocardia and has the ability to decompose L(+)-β-hydroxyisobutyric acid, and the remaining D(-)-β-hydroxyisobutyric acid is decomposed. A method for producing D(-)-β-hydroxyisobutyric acid, which comprises collecting butyric acid. 2 By culturing microorganisms in a medium supplemented with DL(±)-β-hydroxyisobutyric acid, DL
The manufacturing method according to claim 1, wherein (±)-β-hydroxyisobutyric acid and microorganisms are brought into contact. 3 The culture fluid obtained by culturing microorganisms in a nutrient medium
The manufacturing method according to claim 1, wherein the method is made to act on DL(±)-β-hydroxyisobutyric acid. 4.Cultivating microorganisms in a nutrient medium, separating microbial cells from a culture solution to prepare a cell suspension, and applying the suspension to DL(±)-β-hydroxyisobutyric acid. The manufacturing method according to item 1. 5 The microorganisms are Candeida parapsilosis, Candeida rugosa, Hansenula anomala,
Pichia membulanaefaciens, Pichia burtoni, Rhodosporidium toruloides, Sporobolomyces salmonicolar, Sporideoborus jionsonii, Trichosporon
Aquiyuachil, Endomyces obetensis, Endomyces geotrichum, Endomyces lacei, Endomyces tetrasperma, Geotrichum amycelicum, Geotrichum kyandidium, Geotrichum rouvieri, Geotrichum banriei, Mucor alternans, Pecilomyces variotei, Micro The manufacturing method according to claim 1, which is Bacterium luteus, Micrococcus variens, and Nocardia corallina. 6. A patent claim that uses DL(±)-β-hydroxyisobutyric acid alone or in combination with one or more of carbohydrates, alcohols, organic acids, or hydrocarbons that can be assimilated as a carbon source for microorganisms. The manufacturing method according to items 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11559280A JPS5739787A (en) | 1980-08-21 | 1980-08-21 | Preparation of d(-)-beta-hydroxyisobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11559280A JPS5739787A (en) | 1980-08-21 | 1980-08-21 | Preparation of d(-)-beta-hydroxyisobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5739787A JPS5739787A (en) | 1982-03-05 |
| JPH0146112B2 true JPH0146112B2 (en) | 1989-10-05 |
Family
ID=14666413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11559280A Granted JPS5739787A (en) | 1980-08-21 | 1980-08-21 | Preparation of d(-)-beta-hydroxyisobutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5739787A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0089039B1 (en) * | 1982-03-16 | 1987-12-23 | Kanegafuchi Kagaku Kogyo Kabushiki Kaisha | Process for producing d-beta-hydroxyalkanoic acid |
| DK544284A (en) * | 1983-12-06 | 1985-06-07 | Hoffmann La Roche | PROCEDURE FOR THE PREPARATION OF OPTIC ACTIVE PROPIONIC ACID DERIVATIVES |
| US5642953A (en) * | 1993-11-02 | 1997-07-01 | Mitsubishi Pencil Kabushiki Kaisha | Multiplex writing implement |
-
1980
- 1980-08-21 JP JP11559280A patent/JPS5739787A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5739787A (en) | 1982-03-05 |
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