JPH0469993B2 - - Google Patents
Info
- Publication number
- JPH0469993B2 JPH0469993B2 JP62328445A JP32844587A JPH0469993B2 JP H0469993 B2 JPH0469993 B2 JP H0469993B2 JP 62328445 A JP62328445 A JP 62328445A JP 32844587 A JP32844587 A JP 32844587A JP H0469993 B2 JPH0469993 B2 JP H0469993B2
- Authority
- JP
- Japan
- Prior art keywords
- rhodococcus
- weight
- reaction
- nitrile hydratase
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 230000001580 bacterial effect Effects 0.000 claims description 21
- 108010024026 Nitrile hydratase Proteins 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 11
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 11
- -1 amide compound Chemical class 0.000 claims description 11
- WFKAJVHLWXSISD-UHFFFAOYSA-N isobutyramide Chemical compound CC(C)C(N)=O WFKAJVHLWXSISD-UHFFFAOYSA-N 0.000 claims description 11
- 241000187562 Rhodococcus sp. Species 0.000 claims description 10
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 229940047889 isobutyramide Drugs 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 claims description 4
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 4
- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical compound CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 claims description 2
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 claims description 2
- 229940080818 propionamide Drugs 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 3
- 241000187561 Rhodococcus erythropolis Species 0.000 claims 1
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 21
- 239000002609 medium Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 13
- GYCMBHHDWRMZGG-UHFFFAOYSA-N Methylacrylonitrile Chemical compound CC(=C)C#N GYCMBHHDWRMZGG-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 11
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 9
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 7
- 150000001408 amides Chemical class 0.000 description 5
- 238000006703 hydration reaction Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 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 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 235000013372 meat Nutrition 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 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
- 238000004817 gas chromatography Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 229940099596 manganese sulfate Drugs 0.000 description 2
- 239000011702 manganese sulphate Substances 0.000 description 2
- 235000007079 manganese sulphate Nutrition 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-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
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 229940127463 Enzyme Inducers Drugs 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 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
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 150000001323 aldoses Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 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
- 239000004202 carbamide Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Description
（産業上の利用分野）
本発明は、ニトリルヒドラターゼ酵素活性の高
いロドコツカス（Rhodococcus）属菌体を高収
率で生産する方法に関する。
ニトリルヒドラターゼは、ニトリル類を水和し
て対応するアミド類を生成させる酵素として知ら
れており、その産業への利用としては、アクリロ
ニトリルもしくはメタクリロニトリルから、それ
ぞれ対応するアミドへの生成反応が重要であり、
例えば、特開昭62−91189号公報に記載がある。
（従来の技術）
ロドコツカス（Rhodococcus）属に属し、ニ
トリルヒドラターゼを産生する能力を有する細菌
を培養して、ニトリルヒドラターゼ酵素活性を有
する細菌菌体を製造するに当り、特開昭61−
162193号公報には、例えば、ロドコツカスsp.S−
６株の場合に、グルコース、ペプトン、酵母エキ
ス、肉エキスから成る通常の栄養培地で培養し、
活性な菌体を取得していた。
一方、特開昭62−91189号公報には、例えば、
ロドコツカスsp.AK32株の場合には、グルコー
ス、ペプトン、肉エキスなどから成る通常の栄養
培地に、ニトリルを添加した培地を用いて培養
し、より高活性な菌体を取得しており、ニトリル
類がロドコツカスsp.AK32株の酵素誘導物質であ
ることが示されていた。
（発明が解決しようとする問題点）
ニトリルヒドラターゼ酵素活性を有するロドコ
ツカス（Rhodococcus）属細菌の中で、特開昭
62−91189号公報に記載のAK32株、AK33株、
AK3132株は、いずれも誘導酵素を有しており、
高い酵素活性を発現させるためには、さらに簡便
で、かつ有効な酵素誘導物質の開発が望まれてい
た。
（問題点を解決するための手段）
本発明者は、このように、ロドコツカス属細菌
の中で、誘導酵素としてニトリルヒドラターゼを
有している細菌のより高い酵素活性を発現させる
ため、酵素誘導物質およびその使用条件について
鋭意研究を行なつた結果、アミド化合物を培地に
添加して培養することにより、極めて強力なニト
リルヒドラターゼ活性を有するロドコツカス属細
菌を取得することができることを見出し、本発明
を完成するに至つた。
すなわち、本発明は、ロドコツカス属に属し、
ニトリルヒドラターゼを産生する能力を有する細
菌を培養して、ニトリルヒドラターゼ酵素活性を
有する細菌菌体を製造するに当り、アミド化合物
を培地に添加することを特徴とするロドコツカス
属細菌の培養方法に関するものである。
以下、本発明方法は詳細に説明する。
本発明の一般的実施態様としては、ニトリルヒ
ドラターゼを産生する能力を有する細菌を、炭素
源、例えば、グルコース、フラクトース、シユー
クロースおよびアルドースなど、窒素源、例え
ば、硫酸アンモニウム、硝酸アンモニウム、アン
モニアおよび尿素など、有機栄養源、例えば、酵
母エキス、麦芽エキス、肉エキスおよびペプトン
など、無機栄養源、例えば、リン酸塩、ナトリウ
ム、カリウム、鉄、マグネシウム、マンガン、亜
鉛などを適宜含有した培地に、酵素誘導物質とし
て、イソブチルアミド、ｎ−ブチルアミド、プロ
ピオンアミド、アセトアミド、メタクリルアミ
ド、クロトノアミド、アクリルアミドなどのアミ
ド化合物の中の少なくとも一種を添加し培養を行
なう。また、上記酵素誘導物質としてのアミド化
合物を唯一の炭素源としたものに、必要に応じ、
上記の窒素源、無機栄養源および有機栄養源を添
加した培地で培養してもよい。
培地中の該酵素誘導物質の濃度は、通常0.1
ｇ／以上、50ｇ／未満であるが、好ましくは
0.5ｇ／以上、20ｇ／未満となるように調整
する。この濃度が50ｇ／以上になると、菌体増
殖に著しい阻害が見られると共に、取得した菌体
の活性低下が見られる。一方、0.1ｇ／未満で
は、十分に酵素活性を誘導できず、菌体の活性は
低い。培地のPHは、通常５〜９、好ましくは６〜
８、温度は、通常20〜35℃、好ましくは27〜32℃
で、１〜５日間好気的に培養を行なう。
（発明の効果）
本発明にしたがえば、ニトリルヒドラターゼを
産生する能力を有するロドコツカス属細菌に、強
力にニトリルヒドラターゼ酵素活性を発現させる
ことができるため、ニトリルからアミドを酵素法
により製造する際、細菌菌体当りのアミド生産量
を増大させると共に、アミド生産速度を上げるこ
とが可能となり、設備の小型化およびコストの低
減といつた面からの生産性の向上に寄与するとこ
ろが大である。
（実施例）
次に、本発明を実施例により、さらに詳細に説
明するが、本発明の範囲は実施例に限定されるも
のではない。
実施例 １
グルコース２重量％、肉エキス0.1重量％、ペ
プトン0.1重量％、食塩0.1重量％、リン酸第一カ
リウム0.1重量％、硫酸マグネシウム0.05重量％、
硫酸第一鉄0.005重量％、硫酸マンガン0.005重量
％、硫酸アンモニウム0.1重量％、硝酸カリウム
0.1重量％を含んだ培地（以後、Ｚ培地と呼ぶ）
に、イソブチルアミドを0.25重量％添加した後、
水酸化カリウムでPHを7.0に調整したものを、121
℃で30分間滅菌し、室温まで冷却後、ロドコツカ
ス（Rhodococcus）sp.AK33株（微工研菌寄第
1047号）をスラントより一白金耳植菌し、30℃で
38時間培養した。次に、得られた培養液から４℃
で遠心分離により集菌し、0.05Mリン酸バツフア
ー（PH7.0）で洗浄したものを反応に供した。す
なわち、乾燥菌体量として0.2重量％、メタクリ
ルニトリル2.0重量％、0.05Mリン酸バツフアー
（PH7.0）97.8重量％の反応液を調合し、30℃で反
応を開始した。反応開始10分後に、反応液をガス
クロマトグラフにより分析したところ、2.5重量
％のメタクリルアミドを含み、未反応のメタクリ
ロニトリル、メタクリル酸およびその他の副生物
は全く含まれず、反応はほぼ定量的に進行し完結
していた。
実施例 ２〜８
実施例１で用いたＺ培地に、種々のアミド化合
物を0.25重量％添加した後、水酸化カリウムでPH
を7.0に調整したものを、121℃で30分間滅菌し、
室温まで冷却後、ロドコツカス（Rhodococcus）
sp.AK32株（微工研菌寄第1046号）をスラントよ
り一白金耳植菌し、30℃で38時間培養した。次
に、得られた培養液からの集菌、菌体の洗浄およ
びメタクリロニトリルとの水和反応は、実施例１
と同一の方法で行ない、反応時間５分後のメタク
リルアミド収率を比較した。なお、分析にはガス
クロマトグラフイーを用い、得られた結果は第１
表に示した。
(Industrial Application Field) The present invention relates to a method for producing Rhodococcus microbial cells with high nitrile hydratase enzyme activity in high yield. Nitrile hydratase is known as an enzyme that hydrates nitriles to produce the corresponding amides, and its industrial applications include reactions to produce the corresponding amides from acrylonitrile or methacrylonitrile. important,
For example, there is a description in JP-A-62-91189. (Prior Art) In producing bacterial cells having nitrile hydratase enzyme activity by culturing bacteria belonging to the genus Rhodococcus and having the ability to produce nitrile hydratase, JP-A-61
Publication No. 162193 includes, for example, Rhodocotcus sp.S-
In the case of 6 strains, cultured in a normal nutrient medium consisting of glucose, peptone, yeast extract, and meat extract,
Active bacterial cells were obtained. On the other hand, Japanese Patent Application Laid-Open No. 62-91189, for example,
In the case of Rhodococcus sp. AK32 strain, we cultured it in a normal nutrient medium consisting of glucose, peptone, meat extract, etc. and added nitrile to obtain more highly active bacterial cells. was shown to be an enzyme inducer of Rhodococcus sp. AK32 strain. (Problems to be solved by the invention) Among the bacteria of the genus Rhodococcus that have nitrile hydratase enzyme activity,
AK32 strain, AK33 strain described in Publication No. 62-91189,
All AK3132 strains have an inducible enzyme,
In order to express high enzyme activity, it has been desired to develop a simpler and more effective enzyme inducer. (Means for Solving the Problems) The present inventor has developed an enzyme-inducing method in order to express higher enzyme activity in bacteria of the genus Rhodocotcus that have nitrile hydratase as an inducible enzyme. As a result of intensive research on substances and conditions for their use, it was discovered that by adding an amide compound to a culture medium and culturing it, it was possible to obtain bacteria of the genus Rhodocotcus that have extremely strong nitrile hydratase activity, and the present invention I was able to complete it. That is, the present invention belongs to the genus Rhodococcus,
A method for culturing bacteria of the genus Rhodococcus, which comprises adding an amide compound to a medium when culturing bacteria capable of producing nitrile hydratase to produce bacterial cells having nitrile hydratase enzyme activity. It is something. The method of the present invention will be explained in detail below. In a general embodiment of the invention, bacteria capable of producing nitrile hydratase are combined with carbon sources such as glucose, fructose, sucrose and aldose, and nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonia and urea. Enzyme inducers are added to a medium containing organic nutritional sources, such as yeast extract, malt extract, meat extract and peptone, and inorganic nutritional sources, such as phosphate, sodium, potassium, iron, magnesium, manganese, zinc, etc., as appropriate. At least one amide compound such as isobutylamide, n-butylamide, propionamide, acetamide, methacrylamide, crotonamide, acrylamide, etc. is added as a culture. In addition, if necessary, the amide compound as the enzyme inducer is used as the sole carbon source.
It may be cultured in a medium supplemented with the above nitrogen source, inorganic nutrient source, and organic nutrient source. The concentration of the enzyme inducer in the medium is usually 0.1
g/ or more, but less than 50 g/, preferably
Adjust so that it is 0.5g/or more and less than 20g/. When this concentration exceeds 50 g/g, significant inhibition of bacterial cell growth is observed and a decrease in the activity of the obtained bacterial cells is observed. On the other hand, if the amount is less than 0.1 g, enzyme activity cannot be sufficiently induced and the activity of the bacterial cells is low. The pH of the medium is usually 5-9, preferably 6-9.
8. Temperature is usually 20-35℃, preferably 27-32℃
Then, culture is carried out aerobically for 1 to 5 days. (Effects of the Invention) According to the present invention, nitrile hydratase enzyme activity can be strongly expressed in Rhodococcus bacteria that have the ability to produce nitrile hydratase, so that amide can be produced from nitrile by an enzymatic method. In this case, it is possible to increase the amide production amount per bacterial cell and increase the amide production rate, which greatly contributes to improving productivity by downsizing equipment and reducing costs. . (Example) Next, the present invention will be explained in more detail with reference to Examples, but the scope of the present invention is not limited to the Examples. Example 1 Glucose 2% by weight, meat extract 0.1% by weight, peptone 0.1% by weight, salt 0.1% by weight, potassium phosphate 0.1% by weight, magnesium sulfate 0.05% by weight,
Ferrous sulfate 0.005% by weight, manganese sulfate 0.005% by weight, ammonium sulfate 0.1% by weight, potassium nitrate
Medium containing 0.1% by weight (hereinafter referred to as Z medium)
After adding 0.25% by weight of isobutyramide to
121 with pH adjusted to 7.0 with potassium hydroxide
After sterilizing at ℃ for 30 minutes and cooling to room temperature, Rhodococcus sp.
No. 1047) was inoculated from a slant with a platinum loop and grown at 30℃.
Cultured for 38 hours. Next, from the obtained culture solution, 4°C
Bacteria were collected by centrifugation, washed with 0.05M phosphate buffer (PH7.0), and used for reaction. That is, a reaction solution containing 0.2% by weight of dry bacterial cells, 2.0% by weight of methacrylnitrile, and 97.8% by weight of 0.05M phosphate buffer (PH7.0) was prepared, and the reaction was started at 30°C. Ten minutes after the start of the reaction, the reaction solution was analyzed by gas chromatography, and it was found that it contained 2.5% by weight of methacrylamide and no unreacted methacrylonitrile, methacrylic acid, or other by-products, and that the reaction was almost quantitative. It was progressing and completed. Examples 2 to 8 After adding 0.25% by weight of various amide compounds to the Z medium used in Example 1, PH was adjusted with potassium hydroxide.
was adjusted to 7.0 and sterilized at 121℃ for 30 minutes.
After cooling to room temperature, Rhodococcus
A loopful of sp.AK32 strain (Feikoken Bibori No. 1046) was inoculated from a slant and cultured at 30°C for 38 hours. Next, bacterial collection from the obtained culture solution, washing of the bacterial cells, and hydration reaction with methacrylonitrile were carried out in Example 1.
The same method as above was used, and the methacrylamide yields after a reaction time of 5 minutes were compared. In addition, gas chromatography was used for the analysis, and the obtained results are the same as the first one.
Shown in the table.
【表】
実施例 ９〜13
実施例１で用いたＺ培地に、イソブチルアミド
を種々の濃度で添加した後、実施例１と同様な方
法で、ロドコツカスsp.AK32株を植菌し、30℃で
培養を行なつた。菌体の増殖が見られたものにつ
いては、実施例１と同一条件で反応を行ない、反
応時間５分後のメタクリルアミド収率を測定し
た。得られた結果を第２表に示した。[Table] Examples 9 to 13 After adding isobutyramide at various concentrations to the Z medium used in Example 1, Rhodococcus sp. AK32 strain was inoculated in the same manner as in Example 1, and incubated at 30°C. Culture was carried out in For those in which cell growth was observed, the reaction was carried out under the same conditions as in Example 1, and the methacrylamide yield was measured after 5 minutes of reaction time. The results obtained are shown in Table 2.
【表】
実施例 14
リン酸第一カリウム0.1重量％、硫酸マグネシ
ウム0.05重量％、硫酸第一鉄0.005重量％、硫酸
マンガン0.005重量％、硫酸アンモニウム0.1重量
％、硝酸カリウム0.1重量％を含んだ培地（以後
Ｂ培地と呼ぶ）にイソブチルアミドを0.25重量％
添加した後、実施例１と同様な方法で、ロドコツ
カスsp.AK32株を植菌し、30℃で96時間培養し
た。次に、得られた培養液から集菌、菌体の洗浄
およびメタクリロニトリルとの水和反応は、実施
例１と同一の方法で行ない、反応時間15分後に、
反応液を分析したところ、1.2重量％のメタクリ
ルアミドが生成し、1.1重量％のメタクリロニト
リルが検出された。
実施例 15
グルコース１重量％、肉エキス１重量％、ペプ
トン１重量％、食塩0.1重量％を含んだ培地（PH
7.0）を、121℃で30分間滅菌し、室温まで冷却
後、ロドコツカスsp.AK33株をスラントより一白
金耳植菌し、30℃で40時間培養した。次に、得ら
れた培養液から菌体を４℃で遠心分離により集菌
し、生理食塩水で洗浄後、実施例14に示したＢ培
地にイソブチルアミドを0.25重量％添加し、水酸
化カリウムでPH7.0に調整した液に洗浄菌体を投
入し、30℃で８時間撹拌した。次に、この菌体浸
漬液から菌体を分離し、メタクリロニトリルとの
水和反応を実施例１と同一の方法で行ない、反応
時間15分後に反応液を分析したところ、2.1重量
％のメタクリルアミドが生成し、0.3重量％のメ
タクリロニトリルが検出された。
実施例 16
反応基質をメタクリロニトリルからアクリロニ
トリルに変更した以外は、実施例４と同一条件で
アクリロニトリルの水和反応を行ない、反応開始
15分後に、反応液をガスクロマトグラフイーによ
り分析したところ、2.6重量％のアクリルアミド
を含み、未反応のアクリロニトリル、アクリル酸
およびその他の副生物は全く含まれず、反応はほ
ぼ定量的に進行し完結していた。
実施例 17
反応基質をメタクリロニトリルからアクリロニ
トリルに変更した以外は、実施例５と同一条件で
アクリロニトリルの水和反応を行ない、反応開始
15分後に、反応液を分析したところ、2.5重量％
のアクリルアミドが生成し、0.1重量％のアクリ
ロニトリルが検出された。
実施例 18
菌株をロドコツカスsp.AK3132株とし、培養時
間を120時間とした以外は、実施例14と同一条件
で培養した。菌体は、得られた培養液から実施例
１と同様の方法で取得し、反応に供した。すなわ
ち、乾燥菌体量として1.0重量％、メタクリロニ
トリル1.0重量％、0.05Mリン酸バツフアー（PH
7.0）98.0重量％の反応液を調合し、30℃で反応
を開始した。反応開始１時間後に、反応液を分析
したところ、0.4重量％のメタクリルアミドが生
成し、0.7重量％のメタクリロニトリルが検出さ
れた。[Table] Example 14 Medium containing 0.1% by weight of potassium phosphate, 0.05% by weight of magnesium sulfate, 0.005% by weight of ferrous sulfate, 0.005% by weight of manganese sulfate, 0.1% by weight of ammonium sulfate, and 0.1% by weight of potassium nitrate (hereinafter referred to as 0.25% by weight of isobutyramide in (referred to as B medium)
After the addition, Rhodocotcus sp. AK32 strain was inoculated in the same manner as in Example 1, and cultured at 30°C for 96 hours. Next, bacterial collection from the obtained culture solution, washing of the bacterial cells, and hydration reaction with methacrylonitrile were performed in the same manner as in Example 1, and after a reaction time of 15 minutes,
When the reaction solution was analyzed, 1.2% by weight of methacrylamide was produced and 1.1% by weight of methacrylonitrile was detected. Example 15 A medium (PH
7.0) was sterilized at 121°C for 30 minutes, cooled to room temperature, and one platinum loop of Rhodococcus sp. AK33 strain was inoculated from the slant and cultured at 30°C for 40 hours. Next, the bacterial cells were collected from the obtained culture solution by centrifugation at 4°C, and after washing with physiological saline, 0.25% by weight of isobutyramide was added to the B medium shown in Example 14, and potassium hydroxide was added. The washed bacterial cells were added to the solution adjusted to pH 7.0 and stirred at 30°C for 8 hours. Next, the bacterial cells were separated from this bacterial cell immersion liquid, and a hydration reaction with methacrylonitrile was performed in the same manner as in Example 1. After 15 minutes of reaction time, the reaction liquid was analyzed, and it was found that 2.1% by weight. Methacrylamide was formed and 0.3% by weight of methacrylonitrile was detected. Example 16 The hydration reaction of acrylonitrile was carried out under the same conditions as in Example 4, except that the reaction substrate was changed from methacrylonitrile to acrylonitrile, and the reaction was started.
After 15 minutes, the reaction solution was analyzed by gas chromatography and found to contain 2.6% by weight of acrylamide and no unreacted acrylonitrile, acrylic acid, or other by-products, indicating that the reaction proceeded almost quantitatively and was completed. was. Example 17 The hydration reaction of acrylonitrile was carried out under the same conditions as in Example 5, except that the reaction substrate was changed from methacrylonitrile to acrylonitrile, and the reaction was started.
After 15 minutes, the reaction solution was analyzed and found to be 2.5% by weight.
of acrylamide was formed and 0.1% by weight of acrylonitrile was detected. Example 18 The bacterial strain was Rhodococcus sp. AK3132 strain, and the culture was carried out under the same conditions as in Example 14, except that the culture time was 120 hours. Bacterial cells were obtained from the obtained culture solution in the same manner as in Example 1 and subjected to reaction. That is, the dry bacterial mass was 1.0% by weight, methacrylonitrile was 1.0% by weight, and 0.05M phosphate buffer (PH
7.0) A 98.0% by weight reaction solution was prepared and the reaction was started at 30°C. When the reaction solution was analyzed one hour after the start of the reaction, 0.4% by weight of methacrylamide was produced and 0.7% by weight of methacrylonitrile was detected.
Claims (1)
ニトリルヒドラターゼを産生する能力を有する細
菌を培養して、ニトリルヒドラターゼ酵素活性を
有する細菌菌体を製造するに当り、アミド化合物
を培地に添加することを特徴とするロドコツカス
属細菌の培養方法。 ２ アミド化合物がイソブチルアミド、ｎ−ブチ
ルアミド、プロピオンアミド、アセトアミド、メ
タクリルアミド、クロトノアミド、アクリルアミ
ドからなるものである特許請求の範囲第１項記載
の培養方法。 ３ 培地中の該アミド化合物の濃度が0.1ｇ／
以上、50ｇ／未満である特許請求の範囲第１項
記載の培養方法。 ４ ロドコツカス属に属し、ニトリルヒドラター
ゼを産生する能力を有する細菌がロドコツカス
sp.AK32（Rhodococcus sp.AK32）微工研菌寄第
1046号、ロドコツカスsp.AK33（Rhodococcus
sp.AK33）微工研菌寄第1047号またはロドコツカ
ス・エリスロポリスAK3132（Rhodococcus
erythropolis AK3132）微工研菌寄第1040号であ
る特許請求の範囲第１項記載の培養方法。[Claims] 1 Belongs to the genus Rhodococcus,
1. A method for culturing bacteria of the genus Rhodococcus, which comprises adding an amide compound to a medium when culturing bacteria capable of producing nitrile hydratase to produce bacterial cells having nitrile hydratase enzymatic activity. 2. The culture method according to claim 1, wherein the amide compound consists of isobutyramide, n-butyramide, propionamide, acetamide, methacrylamide, crotonamide, and acrylamide. 3 The concentration of the amide compound in the medium is 0.1 g/
The culture method according to claim 1, wherein the amount is less than 50g/. 4 Rhodococcus belongs to the genus Rhodococcus and has the ability to produce nitrile hydratase.
sp.AK32 (Rhodococcus sp.AK32)
No. 1046, Rhodococcus sp. AK33 (Rhodococcus
sp.AK33) Microtechnical Research Institute No. 1047 or Rhodococcus erythropolis AK3132 (Rhodococcus sp.
erythropolis AK3132) The culture method according to claim 1, which is F. erythropolis AK3132).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62328445A JPH01171479A (en) | 1987-12-26 | 1987-12-26 | Method for cultivating bacterium of genus rhodococcus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62328445A JPH01171479A (en) | 1987-12-26 | 1987-12-26 | Method for cultivating bacterium of genus rhodococcus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01171479A JPH01171479A (en) | 1989-07-06 |
| JPH0469993B2 true JPH0469993B2 (en) | 1992-11-09 |
Family
ID=18210353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62328445A Granted JPH01171479A (en) | 1987-12-26 | 1987-12-26 | Method for cultivating bacterium of genus rhodococcus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01171479A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2003033716A1 (en) * | 2001-10-12 | 2005-02-03 | ダイヤニトリックス株式会社 | Method for producing acrylamide and / or methacrylamide using a microbial catalyst |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6143999A (en) * | 1984-08-04 | 1986-03-03 | Koken:Kk | Preparation of acid dispersion liquid containing collagen fiber |
-
1987
- 1987-12-26 JP JP62328445A patent/JPH01171479A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6143999A (en) * | 1984-08-04 | 1986-03-03 | Koken:Kk | Preparation of acid dispersion liquid containing collagen fiber |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01171479A (en) | 1989-07-06 |
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