JP3976355B2 - Process for producing α-hydroxy-4-methylthiobutyric acid - Google Patents
Process for producing α-hydroxy-4-methylthiobutyric acid Download PDFInfo
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- JP3976355B2 JP3976355B2 JP31801494A JP31801494A JP3976355B2 JP 3976355 B2 JP3976355 B2 JP 3976355B2 JP 31801494 A JP31801494 A JP 31801494A JP 31801494 A JP31801494 A JP 31801494A JP 3976355 B2 JP3976355 B2 JP 3976355B2
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- hydroxy
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- methylthiobutyric acid
- methylthiobutyronitrile
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- PICCHNWCTUUCAQ-UHFFFAOYSA-N 2-hydroxypentanethioic s-acid Chemical compound CCCC(O)C(O)=S PICCHNWCTUUCAQ-UHFFFAOYSA-N 0.000 title claims description 17
- 238000000034 method Methods 0.000 title description 9
- 244000005700 microbiome Species 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 11
- VWWOJJANXYSACS-UHFFFAOYSA-N 2-hydroxy-4-methylsulfanylbutanenitrile Chemical compound CSCCC(O)C#N VWWOJJANXYSACS-UHFFFAOYSA-N 0.000 claims description 10
- 230000000813 microbial effect Effects 0.000 claims description 4
- 241000588882 Beijerinckia Species 0.000 claims description 3
- QHDRKFYEGYYIIK-UHFFFAOYSA-N isovaleronitrile Chemical compound CC(C)CC#N QHDRKFYEGYYIIK-UHFFFAOYSA-N 0.000 claims description 3
- ZJAKITOEQQTZLI-UHFFFAOYSA-N 2-methylsulfanylbutanenitrile Chemical compound CCC(SC)C#N ZJAKITOEQQTZLI-UHFFFAOYSA-N 0.000 claims 1
- 241000191844 Pimelia Species 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000002609 medium Substances 0.000 description 9
- 241000186216 Corynebacterium Species 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 229940061720 alpha hydroxy acid Drugs 0.000 description 4
- 150000001280 alpha hydroxy acids Chemical class 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000187619 Actinopolyspora Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzonitrile Chemical compound N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- -1 nitrile compounds Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 241000590020 Achromobacter Species 0.000 description 2
- 241000187844 Actinoplanes Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001467578 Microbacterium Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000588768 Providencia Species 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000187603 Pseudonocardia Species 0.000 description 2
- 241000191025 Rhodobacter Species 0.000 description 2
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- LTYRAPJYLUPLCI-UHFFFAOYSA-N glycolonitrile Chemical compound OCC#N LTYRAPJYLUPLCI-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- WOFDVDFSGLBFAC-UHFFFAOYSA-N lactonitrile Chemical compound CC(O)C#N WOFDVDFSGLBFAC-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000866068 'Nocardioides flavus' Ruan and Zhang 1979 Species 0.000 description 1
- JRHWHSJDIILJAT-BYPYZUCNSA-N (2s)-2-hydroxypentanoic acid Chemical compound CCC[C@H](O)C(O)=O JRHWHSJDIILJAT-BYPYZUCNSA-N 0.000 description 1
- LVRFTAZAXQPQHI-YFKPBYRVSA-N (S)-2-hydroxy-4-methylpentanoic acid Chemical compound CC(C)C[C@H](O)C(O)=O LVRFTAZAXQPQHI-YFKPBYRVSA-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
- MWFMGBPGAXYFAR-UHFFFAOYSA-N 2-hydroxy-2-methylpropanenitrile Chemical compound CC(C)(O)C#N MWFMGBPGAXYFAR-UHFFFAOYSA-N 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000187362 Actinomadura Species 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000186321 Cellulomonas Species 0.000 description 1
- 241000186220 Cellulomonas flavigena Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 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
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241000204057 Kitasatospora Species 0.000 description 1
- 241000204076 Kitasatospora setae Species 0.000 description 1
- 241001249678 Klebsiella pneumoniae subsp. pneumoniae Species 0.000 description 1
- 241001126925 Lobata Species 0.000 description 1
- 241001467566 Microbacterium lacticum Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 241000187580 Nocardioides Species 0.000 description 1
- 241000520272 Pantoea Species 0.000 description 1
- 241000588912 Pantoea agglomerans Species 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- 241000190967 Rhodospirillum Species 0.000 description 1
- 241000190984 Rhodospirillum rubrum Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000187560 Saccharopolyspora Species 0.000 description 1
- 241001134661 Saccharopolyspora rectivirgula Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241001441724 Tetraodontidae Species 0.000 description 1
- 206010048222 Xerosis Diseases 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003674 animal food additive Substances 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
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000909 electrodialysis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- LRDFRRGEGBBSRN-UHFFFAOYSA-N isobutyronitrile Chemical compound CC(C)C#N LRDFRRGEGBBSRN-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003021 water soluble solvent Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
【０００１】
【産業上の利用分野】
本発明はα−ヒドロキシ−４−メチルチオ酪酸の製造法に関する。
【０００２】
α−ヒドロキシ−４−メチルチオ酪酸は、家畜、特に家禽の飼料に含硫アミノ酸類の不足を補う目的で添加される飼料添加物である。
【０００３】
【従来の技術と問題点】
α−ヒドロキシ−４−メチルチオ酪酸の工業的製法としてはα−ヒドロキシ−４−メチルチオブチロニトリルの硫酸による加水分解法が知られている。しかし、硫酸を用いた方法は多量の硫安が副生しα−ヒドロキシ−４−メチルチオ酪酸の回収と精製工程も複雑となる。
【０００４】
一方、最近、ニトリル化合物を微生物の作用により加水分解し対応する酸に変換するいくつかの方法が提案されており、α−ヒドロキシニトリルからのα−ヒドロキシ酸の製造方法に関しては例えば、パチルス属、バクテリジウム属、ミクロコッカス属またはプレビバクテリジウム属の微生物によるラクトニトリルおよびヒドロキシアセトニトリルからの対応する酸の生産［特公昭５８−１５１２０号公報参照］、トルロプシス属酵母による対応するα−ヒドロキシニトリルからの光学活性なＬ−α−ヒドロキシバレリアン酸およびＬ−α−ヒドロキシイソカプロン酸の生産［Fukuda Y.,et al.J.Ferment.Technol. 51 393(1973）参照］、コリネバクテリウム属の微生物を用いたグリコロニトリル、ラクトニトリルおよびアセトンシアンヒドリンの加水分解による対応するα−ヒドロキシ酸の生産 ［特開昭６１−５６０８６号公報参照］およびアルカリゲネス属、シュードモナス属、ロドシュードモナス属、コリネバクテリウム属、アシネトバクター属、バチルス属、マイコバクテリウム属、ロドコッカス属またはキャンデイ ダ属に属する微生物によるα−ヒドロキシニトリルからの光学活性なα−ヒドロキシ酸の生産［特開平２−８４１９８号公報参照］などが示されている。しかし、微生物を用いたα−ヒドロキシ−４−メチルチオ酪酸の製法に関する報告はカセオバクター属、シュードモナス属、アルカリゲネス属、コリネバクテリウム属、ブレビバクテリウム属、ノカルジア属、ロドコッカス属、アースロバクター属の微生物［特開平４−４０８９８号公報参照］のみに示されている。
【０００５】
【問題点を解決するための手段】
本発明者らはこのような従来の製造方法に対し、硫安の生成がなく、エネルギー的にも工業的に有利なα−ヒドロキシ−４−メチルチオ酪酸の製造方法の開発を目的として、α−ヒドロキシ−４−メチルチオブチロニトリルからα−ヒドロキシ−４−メチルチオ酪酸を生産する能力を有する微生物の探索を広範囲に行った結果、ピリメリア（Ｐｉｌｉｍｅｌｉａ）属、及びベイジェリンキア（Ｂｅｉｊｅｒｉｎｃｋｉａ）属に属する微生物に目的とする変換活性を見出し本発明を完成した。
【０００６】
すなわち、本発明は、α−ヒドロキシ−４−メチルチオブチロニトリルをα−ヒドロキシ−４−メチルチオ酪酸に変換させる能力を有する微生物あるいはその処理物をα−ヒドロキシ−４−メチルチオブチロニトリルに作用させ、α−ヒドロキシ−４−メチルチオブチロニトリルをα−ヒドロキシ−４−メチルチオ酪酸に変換させることを特徴とするα−ヒドロキシ−４−メチルチオ酪酸の製造方法である。
【０００７】
本発明で使用する微生物は、ピリメリア（Ｐｉｌｉｍｅｌｉａ）属、又はベイジェリンキア（Ｂｅｉｊｅｒｉｎｃｋｉａ）属に属する菌株であればいずれも使用することが出来るが、具体的な例として例えば、ピリメリア テレバサ（Ｐｉｌｉｍｅｌｉａ ｔｅｒｅｖａｓａ）ＩＦＯ １４５５６、ベイジェリンキア インディカ サブスピシーズ インディカ（Ｂｅｉｊｅｒｉｎｃｋｉａ ｉｎｄｉｃａ ｓｕｂｓｐ． ｉｎｄｉｃａ）ＩＦＯ ３７４４等を挙げることができる。さらに、本発明で使用可能な微生物は、
パントエア（Ｐａｎｔｏｅａ）属、
アクチノマヅラ（Ａｃｔｉｎｏｍａｄｕｒａ）属、
キタサトスポラ（Ｋｉｔａｓａｔｏｓｐｏｒａ）属、
アクロモバクター（Ａｃｈｒｏｍｏｂａｃｔｅｒ）属、
ロモナス（Ｃｅｌｌｕｌｏｍｏｎａｓ）属、
クレブシェラ（Ｋｌｅｂｓｉｅｌｌａ）属、
アクチノポリスポラ（Ａｃｔｉｎｏｐｏｌｙｓｐｏｒａ）属、
アクチノシンネマ（Ａｃｔｉｎｏｓｙｎｎｅｍａ）属、
アクチノプラネス（Ａｃｔｉｎｏｐｌａｎｅｓ）属、
アミコラタ（Ａｍｙｃｏｌａｔａ）属、
サッカロポリスポラ（Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ）属、
ストレプトマイセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属、
ノカルヂオイデス（Ｎｏｃａｒｄｉｏｉｄｅｓ）属、
プロビデンシア（Ｐｒｏｖｉｄｅｎｃｉａ）属、
ミクロバクテリウム（Ｍｉｃｒｏｂａｃｔｅｒｉｕｍ）属、
ロドバクター（Ｒｈｏｄｏｂａｃｔｅｒ）属、又は
ロドスピリリウム（Ｒｈｏｄｏｓｐｉｒｉｌｌｕｍ）属
に属する菌株であればいずれも使用することが出来るが、具体的な例として例えば、パントエア アグロメランス（Ｐａｎｔｏｅａ ａｇｇｌｏｍｍｅｒａｎｓ）ＮＨ−３（ＦＥＲＭ−Ｐ１１３４９）、
アクチノマヅラ クレメーラ サブスピシーズ クレメーラ（Ａｃｔｉｎｏｍａｄｕｒａ ｃｒｅｍｅａ ｓｕｂｓｐ． ｃｒｅｍｅａ）ＩＦＯ １４１８２、
キタサトスポラ セタエ（Ｋｉｔａｓａｔｏｓｐｏｒａ ｓｅｔａｅ）ＩＦＯ１４２１６、
アクロモバクター セロシス（Ａｃｈｒｏｍｏｂａｃｔｅｒ ｘｅｒｏｓｉｓ）ＩＦＯ １２６６８、
セルロモナス フラビジェナ（Ｃｅｌｌｕｌｏｍｏｎａｓ ｆｌａｖｉｇｅｎａ）ＩＦＯ ３７５４、
クレブシェラ フェナウモニエ サブスピシーズ フェナウモニエ（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ ｓｕｂｓｐ． ｐｎｅｕｍｏｎｉａｅ）ＮＨ−３６（ＦＥＲＭ−Ｐ１１７３９）、
アクチノポリスポラ ハロフィラ（Ａｃｔｉｎｏｐｏｌｙｓｐｏｒａ ｈａｌｏｐｈｉｌａ）ＩＦＯ １４１０６、
アクチノシンネマ ミラム（Ａｃｔｉｎｏｓｙｎｎｅｍａ ｍｉｒｕｍ）ＩＦＯ １４０６４、
アクチノプラネス ロバタス（Ａｃｔｉｎｏｐｌａｎｅｓ ｌｏｂａｔｕｓ）ＩＦＯ １２５１３、
アミコラタ アウトトロフィカ（Ａｍｙｃｏｌａｔａ ａｕｔｏｔｒｏｐｈｉｃａ）ＩＦＯ １２７４３、
サッカロポリスポラ レクチヴィルグラ（Ｓａｃｃｈａｒｏｐｏｌｙｓｐｏｒａ ｒｅｃｔｉｖｉｒｇｕｌａ）ＩＦＯ １２１３４、
ストレプトマイセス エスピー（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｓｐ．）ＩＦＯ １３８０９、
ノカルヂオイデス フラバス（Ｎｏｃａｒｄｉｏｉｄｅｓ ｆｌａｖｕｓ）ＩＦＯ １４３９６、
プロビデンシア スタアルティ（Ｐｒｏｖｉｄｅｎｃｉａ ｓｔｕａｒｔｉｉ）ＩＦＯ １２９３０、
ミクロバクテリウム ラクチカム（Ｍｉｃｒｏｂａｃｔｅｒｉｕｍ ｌａｃｔｉｃｕｍ）ＩＦＯ １４１３５、
ロドバクター セフェロイデス（Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈｅｒｏｉｄｅｓ）ＩＦＯ １２２０３、
ロドスピリリウム ルブラム（Ｒｈｏｄｏｓｐｉｒｉｌｌｕｍ ｒｕｂｒｕｍ）ＩＦＯ ３９８６等を挙げることができる。
本発明は、上記微生物の変異株を用いることもできる。これらは公知の微生物であり、発酵研究所（ＩＦＯ）、工業技術院微生物工業技術研究所から容易に入手することができる。
【０００８】
次に本発明の一般的実施態様について説明する。本発明に使用される微生物の培地としては、通常資化し得るグリコース、グリセロール等の炭素源、硫酸アンモニウム、硝酸アンモニウムなどの窒素源、塩化マグネシウム、塩化第二鉄などの無機栄養素を含有する培地か、これらの培地に酵母エキス、肉エキスなどの天然培地を添加したものを用いることができる。誘導源として各々の微生物に適したイソバレロニトリル、イソブチロニトリル、ベンゾニトリルなどのニトリル化合物、またはアセトアミド、プロピオアミドなどのアミド化合物等を用いることもある。
【０００９】
培養条件は、微生物が生育する条件であれば良いが、例えばｐＨ２〜１２、温度５〜５０℃の範囲が望ましく、更に好ましくは好気性条件下でｐＨ４〜１０、温度２０〜５０℃の範囲で選べばよく、培養日数は数時間から１０日程の範囲で活性が最大となるまで培養すればよい。
【００１０】
反応方法は液体培地または平板培地上にて培養した菌体を採取し、必要に応じ固定化菌体、粗酵素、固定化酵素などの菌体処理物を調製し、ｎ−ヘキサン、酢酸エチルなどの適当な溶媒に溶かしたα−ヒドロキシ−４−メチルチオブチロニトリルと水、緩衝液などとの二相系による反応、またはα−ヒドロキシ−４−メチルチオブチロニトリルを水、緩衝液またはエタノール等の水溶性溶媒に溶かして直接菌体または菌体処理物の懸濁液中に混合して行うことができる。
【００１１】
反応条件としては、菌体使用量０．０１〜７０重量％、α−ヒドロキシ−４−メチルチオニトリル濃度０．１〜８０重量％で基質は反応媒体中で完全に溶解しなくてもよい。反応温度は反応が進行する温度であればよいが１〜６０℃、好ましくは５〜４０℃、反応ｐＨは３〜１２、好ましくは６〜１０で５分から１００時間程度反応させればよい。かくしてα−ヒドロキシ−４−メチルチオブチロニトリルは、α−ヒドロキシ−４−メチルチオブチロ酪酸に変換、蓄積される。
【００１２】
生成物の単離は濃縮、イオン交換、電気透析、抽出、晶析などの公知の方法を利用して行うことができる。
【００１３】
【発明の効果】
本発明によれば、α−ヒドロキシ−４−メチルチオニトリルをα−ヒドロキシ−４−メチルチオブチロ酪酸に変換する能力を有する微生物を用いることにより、常温、常圧という温和な条件下で反応を進行させ、副生物として硫安を生成しないα−ヒドロキシ−４−メチルチオ酪酸の工業的に有利な製法を提供することができる。
【００１４】
次に、本発明を実施例によりさらに詳細に説明するが、本発明はこれら実施例に限定されるものではない。
【００１５】
【実施例１】
（Ａ）培養
表１に示す菌体を下記により培養した。
【００１６】
【００１７】
２）培養条件
斜面培地から１白金耳の菌体を採り、上記液体培地６ｍｌ／２１ｍｍ試験管に接種し、３０℃で４８時間好気条件下に振盪培養した。
【００１８】
（Ｂ）反応
液体培地から菌体を遠心分離により集菌して各々の菌体を０．０５リン酸緩衝液（ｐＨ７．０）で３回洗浄した。菌体を1.5 ｍｌの同様の緩衝液に再懸濁し終濃度５０ｍＭのα−ヒドロキシ−４−メチルチオブチロニトリルを添加し３０℃で２４時間、振盪しながら反応を行った。反応終了後、各々の反応液を遠心分離し菌体を除去し、遠心上清中のα−ヒドロキシ酸は液体クロマトグラフィー（カラム；UNISIL-PAC 5C18-250A、キャリア；０．１％リン酸：アセトニトリル＝９：１、カラム温度；４０℃、検出；２１０ｎｍ）により定量した。
【００１９】
結果を表１に示した。
【００２０】
【表１】
[Industrial application fields]
The present invention relates to a process for producing α-hydroxy-4-methylthiobutyric acid.
[0002]
α-Hydroxy-4-methylthiobutyric acid is a feed additive that is added to livestock, particularly poultry feed, to compensate for the shortage of sulfur-containing amino acids.
[0003]
[Conventional technology and problems]
As an industrial production method of α-hydroxy-4-methylthiobutyric acid, a hydrolysis method of α-hydroxy-4-methylthiobutyronitrile with sulfuric acid is known. However, in the method using sulfuric acid, a large amount of ammonium sulfate is by-produced, and the recovery and purification process of α-hydroxy-4-methylthiobutyric acid is complicated.
[0004]
On the other hand, recently, several methods for hydrolyzing nitrile compounds by the action of microorganisms and converting them into corresponding acids have been proposed. Regarding the method for producing α-hydroxy acids from α-hydroxynitriles, for example, Production of the corresponding acid from lactonitrile and hydroxyacetonitrile by microorganisms of the genus Bacterium, Micrococcus or Plebacteria [see Japanese Patent Publication No. 58-15120], from the corresponding α-hydroxynitrile by Toluropsis yeast Production of optically active L-α-hydroxyvaleric acid and L-α-hydroxyisocaproic acid (see Fukuda Y., et al. J. Ferment. Technol. 51 393 (1973)), microorganisms belonging to the genus Corynebacterium By hydrolysis of the glycolonitrile, lactonitrile and acetone cyanohydrin used. Production of corresponding α-hydroxy acids [see JP-A-61-56086] and Alkaligenes, Pseudomonas, Rhodopseudomonas, Corynebacterium, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus or Candy Production of optically active α-hydroxy acids from α-hydroxynitriles by microorganisms belonging to the genus Da (see JP-A-2-84198) and the like are shown. However, reports on the production method of α-hydroxy-4-methylthiobutyric acid using microorganisms include microorganisms belonging to the genus Caseobacter, Pseudomonas, Alkaligenes, Corynebacterium, Brevibacterium, Nocardia, Rhodococcus, and Arthrobacter [See Japanese Patent Laid-Open No. 4-40898] only.
[0005]
[Means for solving problems]
For the purpose of developing an α-hydroxy-4-methylthiobutyric acid production method that does not produce ammonium sulfate and is industrially advantageous from the standpoint of such a conventional production method, the present inventors have developed α-hydroxy. As a result of extensive search for microorganisms having the ability to produce α-hydroxy-4-methylthiobutyric acid from -4-methylthiobutyronitrile , it was found that the microorganisms belong to the genus Pirimelia and the genus Beijerinckia. The target conversion activity was found and the present invention was completed.
[0006]
That is, the present invention allows a microorganism having the ability to convert α-hydroxy-4-methylthiobutyronitrile to α-hydroxy-4-methylthiobutyric acid or a processed product thereof to act on α-hydroxy-4-methylthiobutyronitrile. , Α-hydroxy-4-methylthiobutyronitrile is converted to α-hydroxy-4-methylthiobutyric acid, which is a method for producing α-hydroxy-4-methylthiobutyric acid.
[0007]
Any microorganism can be used as the microorganism used in the present invention as long as it belongs to the genus Pimeria, or the genus Beijerinckia. Specific examples include, for example, Pimeria terevasa. IFO 14556, Beigerinkia indica subspices Indica IFO 3744, and the like. Furthermore, microorganisms that can be used in the present invention are:
Pantoea genus,
Actinomadura genus,
Kitasatospora genus ,
Achromobacter genus ,
Genus of Cellulomonas,
Klebsiella genus,
Actinopolyspora (Actinopolyspora),
Actinosinnema genus,
Actinoplanes genus,
Genus Amycolata,
Saccharopolyspora genus,
The genus Streptomyces,
Nocardioides genus,
Providencia genus,
Microbacterium (genus Microbacterium),
Any strain can be used as long as it belongs to the genus Rhodobacter or Rhodospirillum. For example, Pantoea agglomerans NH-3 (FERM-P11349) ,
Actinomadera Cremera Subspices Cremera (Actinoma cremea subsp. Cremea) IFO 14182,
Kitasatospora setae IFO 14216 ,
Achromobacter xerosis IFO 12668 ,
Cellulomonas flavigena IFO 3754,
Klebsiella fenaumonie subspice phenaumonie (Klebsiella pneumoniae subsp. Pneumoniae) NH-36 (FERM-P11739),
Actinopolyspora halophylla IFO 14106,
Actinosynema mirum IFO 14064,
Actinoplanes lobatas IFO 12513,
Amycolata autotropica IFO 12743,
Saccharopolyspora rectivirgula IFO 12134,
Streptomyces sp. IFO 13809,
Nocardioides flavus IFO 14396,
Providencia staarti IFO 12930,
Microbacterium lacticum IFO 14135,
Rhodobacter spheroides IFO 12203,
Examples thereof include Rhodospirillum rubrum IFO 3986.
In the present invention, a mutant strain of the above microorganism can also be used. These are known microorganisms and can be easily obtained from the Fermentation Research Institute (IFO) and the National Institute of Microbiology and Industrial Technology.
[0008]
Next, general embodiments of the present invention will be described. As a microorganism culture medium used in the present invention, a medium containing carbon sources such as glucose, glycerol and the like, nitrogen sources such as ammonium sulfate and ammonium nitrate, and inorganic nutrients such as magnesium chloride and ferric chloride, or these A medium obtained by adding a natural medium such as yeast extract or meat extract to the above medium can be used. Nitrile compounds such as isovaleronitrile, isobutyronitrile, and benzonitrile suitable for each microorganism, or amide compounds such as acetamide and propioamide may be used as the induction source.
[0009]
The culture conditions may be any conditions that allow microorganisms to grow. For example, a pH range of 2 to 12 and a temperature range of 5 to 50 ° C. are desirable, and more preferably a pH range of 4 to 10 and a temperature range of 20 to 50 ° C. under aerobic conditions. What is necessary is just to choose, and what is necessary is just to culture | cultivate until activity becomes the maximum in the range for several days to about 10 days.
[0010]
The reaction method is to collect microbial cells cultured on a liquid medium or flat plate medium, and prepare microbial cell processed products such as immobilized microbial cells, crude enzyme, and immobilized enzyme as necessary, such as n-hexane, ethyl acetate, etc. A reaction in a two-phase system of α-hydroxy-4-methylthiobutyronitrile and water, buffer, etc. dissolved in a suitable solvent, or α-hydroxy-4-methylthiobutyronitrile in water, buffer, ethanol, etc. In a water-soluble solvent, and directly mixed into a suspension of cells or treated cells.
[0011]
As reaction conditions, the amount of cells used is 0.01 to 70% by weight, the α-hydroxy-4-methylthionitrile concentration is 0.1 to 80% by weight, and the substrate may not be completely dissolved in the reaction medium. The reaction temperature may be any temperature at which the reaction proceeds, but it may be performed at 1 to 60 ° C., preferably 5 to 40 ° C., and the reaction pH is 3 to 12, preferably 6 to 10, for 5 minutes to 100 hours. Thus, α-hydroxy-4-methylthiobutyronitrile is converted and accumulated into α-hydroxy-4-methylthiobutyrobutyric acid.
[0012]
The product can be isolated using a known method such as concentration, ion exchange, electrodialysis, extraction or crystallization.
[0013]
【The invention's effect】
According to the present invention, by using a microorganism having the ability to convert α-hydroxy-4-methylthionitrile to α-hydroxy-4-methylthiobutyrobutyric acid, the reaction proceeds under mild conditions of normal temperature and normal pressure. Thus, an industrially advantageous process for producing α-hydroxy-4-methylthiobutyric acid that does not produce ammonium sulfate as a by-product can be provided.
[0014]
EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
[0015]
[Example 1]
(A) Culture The cells shown in Table 1 were cultured as follows.
[0016]
[0017]
2) Culture conditions One platinum loop of fungus was taken from the slant medium, inoculated into the above 6 ml / 21 mm test tube of liquid medium, and cultured with shaking at 30 ° C. for 48 hours under aerobic conditions.
[0018]
(B) Cells were collected from the reaction liquid medium by centrifugation, and each cell was washed 3 times with 0.05 phosphate buffer (pH 7.0). The cells were resuspended in 1.5 ml of the same buffer, α-hydroxy-4-methylthiobutyronitrile having a final concentration of 50 mM was added, and the reaction was carried out at 30 ° C. with shaking for 24 hours. After completion of the reaction, each reaction solution is centrifuged to remove the cells, and α-hydroxy acid in the supernatant is subjected to liquid chromatography (column; UNISIL-PAC 5C18-250A, carrier; 0.1% phosphoric acid: Acetonitrile = 9: 1, column temperature; 40 ° C., detection; 210 nm).
[0019]
The results are shown in Table 1.
[0020]
[Table 1]
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31801494A JP3976355B2 (en) | 1994-12-21 | 1994-12-21 | Process for producing α-hydroxy-4-methylthiobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31801494A JP3976355B2 (en) | 1994-12-21 | 1994-12-21 | Process for producing α-hydroxy-4-methylthiobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH08173175A JPH08173175A (en) | 1996-07-09 |
| JP3976355B2 true JP3976355B2 (en) | 2007-09-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| JP31801494A Expired - Lifetime JP3976355B2 (en) | 1994-12-21 | 1994-12-21 | Process for producing α-hydroxy-4-methylthiobutyric acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE69737696T2 (en) * | 1996-02-29 | 2008-01-10 | Nippon Soda Co. Ltd. | PROCESS FOR THE PREPARATION OF ALPHA HYDROXYLIC ACIDS WITH THE HELP OF MICROORGANISM AND NEW MICROORGANISM. |
| JPH10179183A (en) * | 1996-12-20 | 1998-07-07 | Daicel Chem Ind Ltd | Method for producing carboxylic acid |
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1994
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| Publication number | Publication date |
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| JPH08173175A (en) | 1996-07-09 |
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