JPH01171478A - Method for cultivating bacterium of genus rhodococcus - Google Patents
Method for cultivating bacterium of genus rhodococcusInfo
- Publication number
- JPH01171478A JPH01171478A JP62328444A JP32844487A JPH01171478A JP H01171478 A JPH01171478 A JP H01171478A JP 62328444 A JP62328444 A JP 62328444A JP 32844487 A JP32844487 A JP 32844487A JP H01171478 A JPH01171478 A JP H01171478A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- rhodococcus
- nitrile hydratase
- weight
- reaction
- 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
- 241000316848 Rhodococcus <scale insect> Species 0.000 title claims abstract description 21
- 241000894006 Bacteria Species 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 6
- 108010024026 Nitrile hydratase Proteins 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 14
- KQNPFQTWMSNSAP-UHFFFAOYSA-N isobutyric acid Chemical compound CC(C)C(O)=O KQNPFQTWMSNSAP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000007524 organic acids Chemical class 0.000 claims abstract description 12
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims abstract description 6
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims abstract description 6
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 claims abstract description 3
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 claims abstract description 3
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims abstract description 3
- 235000019260 propionic acid Nutrition 0.000 claims abstract description 3
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims abstract description 3
- 238000012258 culturing Methods 0.000 claims description 9
- 241000187562 Rhodococcus sp. Species 0.000 claims description 5
- 238000012136 culture method Methods 0.000 claims 2
- 241000187561 Rhodococcus erythropolis Species 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 229940005605 valeric acid Drugs 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 description 22
- 239000002609 medium Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- GYCMBHHDWRMZGG-UHFFFAOYSA-N Methylacrylonitrile Chemical compound CC(=C)C#N GYCMBHHDWRMZGG-UHFFFAOYSA-N 0.000 description 10
- 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
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-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 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 150000001408 amides Chemical class 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000006703 hydration reaction Methods 0.000 description 5
- 235000013372 meat Nutrition 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-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
- 239000000411 inducer Substances 0.000 description 4
- 238000004519 manufacturing process Methods 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
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 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
- 230000002255 enzymatic effect Effects 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
- 239000000203 mixture Substances 0.000 description 2
- 150000002825 nitriles Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 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
- 150000003839 salts Chemical class 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 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
- 241001478240 Coccus Species 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
- 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
- 238000004140 cleaning Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- -1 hydrates nitriles Chemical class 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
- 235000016709 nutrition Nutrition 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
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000005406 washing Methods 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
- Enzymes And Modification Thereof (AREA)
- 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】
（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
ッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）属菌体を高収率で生
産する方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing Rhodococcus cells with high nitrile hydratase enzyme activity in high yield.
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。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. This is important and is described in, for example, Japanese Patent Application Laid-Open No. 62-91189.
（従来の技術）
ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）属に属し、ニ
トリルヒドラターゼを産生ず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、ロドコッカス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
。(Prior Art) In the production of 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-61-162193 In the publication, for example, in the case of Rhodococcus sp. S-6 strain, active bacterial cells were obtained by culturing in a conventional nutrient medium consisting of glucose, peptone, yeast extract, and meat extract.
一方、特開昭６２−９１１８９号公報には、例えば、ロ
ドコフカス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ッカスｓｐ、Ａ
Ｋ３２株の酵素誘導物質であることが示されていた。On the other hand, in JP-A-62-91189, for example, in the case of Rhodocovcus sp, AK32 strain, glucose,
Bacteria with higher activity are obtained by culturing in a medium containing nitrile in addition to a normal nutrient medium consisting of peptone, meat extract, etc.
It was shown to be an enzyme inducer of the K32 strain.
、（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ッカス（
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。, (Problem to be solved by the invention) Rhodococcus having nitrile hydratase enzyme activity (
Among the bacteria of the genus Rhodococcus, JP-A-62-
AK32 strain, 'AK33 strain, described in Publication No. 91189,
All of the AK3132 strains have an inducing enzyme, and in order to express high enzyme activity, it has been desired to develop a simpler and more effective enzyme inducing substance.
（問題点を解決するための手段）
本発明者は、このように、ロドコッカス属細菌の中で、
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った結果、有
機酸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ッカス属細
菌を取得することができることを見出し、本発明を完成
するに至った。(Means for solving the problem) The present inventor has thus discovered that among Rhodococcus bacteria,
In order to express higher enzymatic activity in bacteria that have nitrile hydratase as an inducing enzyme, we conducted extensive research on enzyme inducers and conditions for their use, and found that by adding organic acids to the culture medium and culturing, The inventors have discovered that it is possible to obtain bacteria of the genus Rhodococcus that have extremely strong nitrile hydratase activity, and have completed the present invention.
すなわち、本発明は、ロドコッカス属に属し、ニトリル
ヒドラターゼを産生ず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、有機酸を培地に添加することを特徴とする
ロドコッカス属細菌の培養方法に関するものである。That is, the present invention involves culturing a bacterium belonging to the genus Rhodococcus and having the ability to produce nitrile hydratase,
The present invention relates to a method for culturing bacteria of the genus Rhodococcus, which comprises adding an organic acid to a medium in producing bacterial cells having nitrile hydratase enzyme activity.
以下、本発明方法は詳細に説明する。The method of the present invention will be explained in detail below.
本発明の一般的実施態様としては、ニトリルヒドラター
ゼを産生ず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、シュ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、プロピオン
酸、ｎ−酪酸、イソ酪酸、ｎ−吉草酸、イソ吉草酸など
の有機酸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有機酸を唯一の炭素
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。In a general embodiment of the invention, bacteria capable of producing nitrile hydratases are combined with carbon sources such as glucose, fructose, sucrose and aldose, and nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonia and urea. , organic nutritional sources,
For example, propionic acid is added as an enzyme inducer to a medium containing appropriate inorganic nutrients such as yeast extract, malt extract, meat extract, and peptone, such as phosphate, sodium, potassium, iron, magnesium, manganese, and zinc. , n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, and the like are added and cultured. Alternatively, the culture may be carried out in a medium in which the organic acid as the enzyme inducer is the only carbon source, and the nitrogen source, inorganic nutrient source, and organic nutrient source described above are added as necessary.
培地中の該酵素誘導物質の濃度は、通常０．１ｇ／１以
上、２０ｇ／β未満であるが、好ましくは０．５ｇ／１
以上、５ｇ／１未満となるように調整する。この濃度が
２０ ｇ／ｌ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、０．１ ｇ ／ １２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。The concentration of the enzyme inducer in the medium is usually 0.1 g/1 or more and less than 20 g/β, preferably 0.5 g/1.
The above is adjusted so that it is less than 5 g/1. When this concentration exceeds 20 g/l, a 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 it is less than 0.1 g/12, 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 6-8, temperature usually 20-35°C,
The culture is preferably carried out aerobically at 27-32°C for 1-5 days.
（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ずる
能力を有するロドコッカス属細菌に、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
トの低減といった面からの生産性の向上に寄与するとこ
ろが大である。(Effects of the Invention) According to the present invention, it is possible to make Rhodococcus bacteria capable of producing nitrile hydratase strongly express nitrile hydratase enzymatic activity, so that amide can be produced from nitrile by an enzymatic method. In this case, it becomes possible to increase the amide production amount per bacterial cell and to increase the amide production rate, which greatly contributes to improving productivity in terms of downsizing of equipment and cost reduction.
（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。(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.
実施例１
グルコース２重量％、肉エキス０．１ｆｆｉ１％、ペプ
トン０．１重量％、食塩０．１重量％、リン酸第−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． ＯＯ５重量％、硫酸マンガン０、００
５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２培地と呼ぶ）に、
イソ酪酸を０．２５重量％添加した後、水酸化カリウム
でｐ［（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、ロドコッカス（Ｒｈｏｄｏ
ｃｏｃｃｕｓ） ｓｐ、 Ａ Ｋ ３３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リン酸バッファー（
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、タクリロニトリ
ル２．０重量％、０．０５Ｍリン酸バッファー（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１５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。Example 1 Glucose 2% by weight, meat extract 0.1ffi1%, 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. OO5% by weight, manganese sulfate 0.00
In a medium containing 5% by weight, 0.1% by weight of ammonium sulfate, and 0.1% by weight of potassium nitrate (hereinafter referred to as 2 medium),
After adding 0.25% by weight of isobutyric acid, p
coccus) sp, A K 33 strain (Feikoken Bacteria No. 1047) was inoculated from a slant into a platinum loop and kept at 30°C.
The cells were cultured for 38 hours. Next, bacteria were collected from the obtained culture solution by centrifugation at 4°C, and 0.05M phosphate buffer (
pH 7.0) was used for reaction. That is, 0.2% by weight of dry bacterial cells, 2.0% by weight of methacrylonitrile, and 0.05M phosphate buffer (pH 7).
, 0) A 97.8% by weight reaction solution was prepared, and the reaction was started at 30°C. When the reaction solution was analyzed by gas chromatography 15 minutes after the start of the reaction, 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 progressed and was completed.
実施例２〜６
実施例１で用いたＺ培地に、種々の有機酸を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、ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ）ｓｐ、Ａ
Ｋ３２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。ただし、有
機酸がメタクリル酸とアクリル酸の場合には、ｐＨを７
．０に調整したＺ培地を、先ず滅菌し、冷却後、メタク
リル酸またはアクリル酸と、ｐＨｌ整に必要な量の水酸
化カリウムを無菌的に添加したものを用い、３０℃で１
３３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
マトグラフィーを用い、得られた結果は第１表に示した
。Examples 2 to 6 After adding 0.25% by weight of various organic acids to the Z medium used in Example 1, the pH was adjusted to 7.0 with potassium hydroxide, and the mixture was sterilized at 121°C for 30 minutes. After cooling to room temperature, Rhodococcus sp, A
One platinum loop of K32 strain (Feikoken Bibori No. 1046) was inoculated from a slant and cultured at 30°C for 38 hours. However, if the organic acids are methacrylic acid and acrylic acid, the pH should be adjusted to 7.
．． Z medium adjusted to 0 was first sterilized, cooled, and then sterilized at 30°C using methacrylic acid or acrylic acid and potassium hydroxide in an amount necessary to adjust the pH.
Cultured for 33 hours. Next, collect bacteria from the obtained culture solution,
Cleaning of bacterial cells and hydration reaction with methacrylonitrile are
This was carried out in the same manner as in Example 1, and the methacrylamide yields after a reaction time of 5 minutes were compared. Note that gas chromatography was used for the analysis, and the obtained results are shown in Table 1.
第 １ 表
実施例７〜９
実施例１で用いたＺ培地に、イソ酪酸を種々の濃度で添
加した後、実施例１と同様な方法で、ロドコッカスｓｐ
、ＡＫ３２株を植菌し、３０℃で培養を行なった。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。Table 1 Examples 7 to 9 After adding isobutyric acid at various concentrations to the Z medium used in Example 1, Rhodococcus sp.
, AK32 strain was inoculated and cultured at 30°C. 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.
得られた結果を第２表に示した。The results obtained are shown in Table 2.
第 ２ 表
実施例１０
リン酸第−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ｏ、ｏｏｓ重量％、硫酸マン
ガンｏ、ｏｏｓ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）にイソ酪酸を０．２５重量％添加した後、実施
例１と同様な方法で、ロドコッカス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１５分後に、反応液を分析したところ、反応はほぼ
定量的に進行し、２．５重量％のメタクリルアミドが生
成していた。Table 2 Example 10 Potassium phosphate 0.1% by weight, magnesium sulfate 0
．． 05 wt%, ferrous sulfate o, oos wt%, manganese sulfate o, oos wt%, ammonium sulfate 0.1 wt%
After adding 0.25% by weight of isobutyric acid to a medium containing 0.1% by weight of potassium nitrate (hereinafter referred to as B medium), Rhodococcus sp, AK32 strain was inoculated in the same manner as in Example 1, The cells were 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 15 minutes of reaction time, the reaction solution was analyzed. The reaction proceeded almost quantitatively, producing 2.5% by weight of methacrylamide.
実施例１１
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（ｐ）Ｉ７．０）
を、１２１℃で３０分間滅菌し、室温まで冷却後、ロド
コッカス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。Example 11 Medium containing 1% by weight of glucose, 1% by weight of meat extract, 1% by weight of peptone, and 0.1% by weight of salt (p)I7.0)
was sterilized at 121°C for 30 minutes, cooled to room temperature, and Rhodococcus sp, AK33 strain was inoculated from a slant using a loopful and cultured at 30°C for 40 hours.
次に、得られた培養液から菌体を４℃下で遠心分離によ
り集菌し、生理食塩水で洗浄後、実施例１０に示したＢ
培地に、イソ酪酸を０．２５重量％添加し、水酸化カリ
ウムでｐｏ’ｒ、ｏに調整した液に洗浄菌体を投入し、
３０℃で８時間攪拌した。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、１．９重量％のメタクリルアミドが生
成し、０．５重量％のメタクリロニトリルが検出された
。Next, the bacterial cells were collected from the obtained culture solution by centrifugation at 4°C, washed with physiological saline, and then
0.25% by weight of isobutyric acid was added to the culture medium, and the washed bacterial cells were added to a solution adjusted to po'r and o with potassium hydroxide,
The mixture was 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 the weight was 1.9. % of methacrylamide was formed and 0.5% by weight of methacrylonitrile was detected.
実施例１２
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２０分後に、反応液を
ガスクロマトグラフィ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。Example 12 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 20 minutes after the start of the reaction, the reaction solution was analyzed by gas chromatography. .6
It contained % by weight of acrylamide and no unreacted acrylonitrile, acrylic acid, or other by-products, and the reaction proceeded almost quantitatively to completion.
実施例１３
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２０分後に、反応液を
分析したところ、０．４重量％のアクリルアミドが生成
し、１．７重量％のアクリロニトリルが検出された。Example 13 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. Twenty minutes after the start of the reaction, the reaction solution was analyzed and found to be 0.4% by weight. of acrylamide was produced and 1.7% by weight of acrylonitrile was detected.
実施例１４
菌株をロドコッカスｓｐ、ＡＫ ３１３２株とし、培養
時間を１２０時間とした以外は、実施例１０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％、メタクリコニトリル１．０重量
％、０．０５Ｍリン酸バッファー（ｐＨ７，０） 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．
３重量％のメタクリルアミドが生成し、０．８重量％の
メタクリロニトリルが検出された。Example 14 The strain was Rhodococcus sp, AK 3132 strain, and culture was performed under the same conditions as in Example 10, 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, 1.0% by weight of dry bacterial cells, 1.0% by weight of methacriconitrile, 0.05M phosphate buffer (pH 7.0) 98
．． A 0% by weight reaction solution was prepared, and the reaction was started at 30°C. One hour after the start of the reaction, the reaction solution was analyzed and found to be 0.
3% by weight of methacrylamide was produced and 0.8% by weight of methacrylonitrile was detected.
Claims (4)
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、有機酸を培地に添加すること
を特徴とするロドコッカス属細菌の培養方法。(1) Adding an organic acid to the medium when culturing bacteria belonging to the genus Rhodococcus and having the ability to produce nitrile hydratase to produce bacterial cells having nitrile hydratase enzyme activity. A method for culturing Rhodococcus bacteria, characterized by:
−吉草酸、イソ吉草酸から選ばれた化合物である特許請
求の範囲第１項記載の培養方法。(2) The organic acid is propionic acid, n-butyric acid, isobutyric acid, n-
- The culturing method according to claim 1, wherein the compound is selected from valeric acid and isovaleric acid.
ｇ／ｌ未満である特許請求の範囲第１項記載の培養方法
。(3) The concentration of organic acid in the medium is 0.1 g/l or more, 20
The culture method according to claim 1, wherein the concentration is less than g/l.
産生する能力を有する細菌がロドコッカスｓｐ．ＡＫ３
２（Ｒｈｏｄｏｃｏｃｃｕｓ ｓｐ．ＡＫ３２）微工研
菌寄第１０４６号、ロドコッカスｓｐ．ＡＫ３３（Ｒｈ
ｏｄｏｃｏｃｃｕｓ ｓｐ．ＡＫ３３）微工研菌寄第１
０４７号またはロドコッカス・エリスロポリスＡＫ３１
３２（Ｒｈｏｄｏｃｏｃｃｕｓ ｅｒｙｔｈｒｏｐｏｌ
ｉｓ 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。(4) Bacteria belonging to the genus Rhodococcus and having the ability to produce nitrile hydratase are Rhodococcus sp. AK3
2 (Rhodococcus sp. AK32) Microtechnical Research Institute No. 1046, Rhodococcus sp. AK33 (Rh
odococcus sp. AK33) Microtechnical Research Institute 1st
No. 047 or Rhodococcus erythropolis AK31
32 (Rhodococcus erythropol
is AK3132) The culture method according to claim 1, which is AK3132).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62328444A JPH01171478A (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 |
|---|---|---|---|
| JP62328444A JPH01171478A (en) | 1987-12-26 | 1987-12-26 | Method for cultivating bacterium of genus rhodococcus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01171478A true JPH01171478A (en) | 1989-07-06 |
| JPH0469992B2 JPH0469992B2 (en) | 1992-11-09 |
Family
ID=18210342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62328444A Granted JPH01171478A (en) | 1987-12-26 | 1987-12-26 | Method for cultivating bacterium of genus rhodococcus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01171478A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6350381B2 (en) * | 1998-10-27 | 2002-02-26 | Kinder Morgan Energy Partners, L.P. | Biodegradation of ethers using fatty acid enhanced microbes |
| JP2017529849A (en) * | 2014-09-30 | 2017-10-12 | ビーエーエスエフ ソシエタス・ヨーロピアＢａｓｆ Ｓｅ | Method for culturing microorganisms having nitrile hydratase activity |
-
1987
- 1987-12-26 JP JP62328444A patent/JPH01171478A/en active Granted
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6350381B2 (en) * | 1998-10-27 | 2002-02-26 | Kinder Morgan Energy Partners, L.P. | Biodegradation of ethers using fatty acid enhanced microbes |
| JP2017529849A (en) * | 2014-09-30 | 2017-10-12 | ビーエーエスエフ ソシエタス・ヨーロピアＢａｓｆ Ｓｅ | Method for culturing microorganisms having nitrile hydratase activity |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0469992B2 (en) | 1992-11-09 |
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