JPH04320680A - Culture of bacterium belonging to genus pseudomonas, capable of forming indigo - Google Patents
Culture of bacterium belonging to genus pseudomonas, capable of forming indigoInfo
- Publication number
- JPH04320680A JPH04320680A JP11077791A JP11077791A JPH04320680A JP H04320680 A JPH04320680 A JP H04320680A JP 11077791 A JP11077791 A JP 11077791A JP 11077791 A JP11077791 A JP 11077791A JP H04320680 A JPH04320680 A JP H04320680A
- Authority
- JP
- Japan
- Prior art keywords
- indigo
- culture
- salt
- benzoic acid
- cells
- 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.)
- Pending
Links
- 235000000177 Indigofera tinctoria Nutrition 0.000 title claims abstract description 31
- 229940097275 indigo Drugs 0.000 title claims abstract description 31
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 241000589516 Pseudomonas Species 0.000 title claims description 8
- 241000894006 Bacteria Species 0.000 title abstract description 9
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000005711 Benzoic acid Substances 0.000 claims abstract description 12
- 235000010233 benzoic acid Nutrition 0.000 claims abstract description 12
- 150000003839 salts Chemical class 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 244000005700 microbiome Species 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 abstract description 7
- 239000004299 sodium benzoate Substances 0.000 abstract description 7
- 235000010234 sodium benzoate Nutrition 0.000 abstract description 7
- 239000000243 solution Substances 0.000 abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 4
- 241000589774 Pseudomonas sp. Species 0.000 abstract description 4
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 abstract description 4
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 abstract description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 239000000725 suspension Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 13
- 238000012136 culture method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N Arginine Chemical compound OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 108010032438 4-xylene methylhydroxylase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 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
- 238000003794 Gram staining Methods 0.000 description 1
- 229910004861 K2 HPO4 Inorganic materials 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-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
- 229940000635 beta-alanine Drugs 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940110377 dl- arginine Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000011090 malic acid Nutrition 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
- 239000000203 mixture Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 238000003541 multi-stage reaction Methods 0.000 description 1
- 108010041042 naphthalene dioxygenase Proteins 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 229940088594 vitamin Drugs 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
- 239000012138 yeast extract Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、インジゴ生成能を有す
るシュードモナス属に属する微生物の培養方法に関し、
さらに詳しくは、該微生物のインジゴ生成能及び菌体収
量を向上させる培養方法に関する。[Field of Industrial Application] The present invention relates to a method for culturing microorganisms belonging to the genus Pseudomonas that have the ability to produce indigo.
More specifically, the present invention relates to a culture method for improving the indigo-producing ability and cell yield of the microorganism.
【０００２】0002
【従来の技術】従来、インジゴは工業用染料として広く
利用されており、化学合成法により製造されている。し
かしながら、化学合成法は、反応が多段階になるので収
率が悪く、また化学的分解による副生物が多い等の欠点
があった。BACKGROUND OF THE INVENTION Hitherto, indigo has been widely used as an industrial dye, and is produced by chemical synthesis. However, chemical synthesis methods have drawbacks such as poor yields due to multi-step reactions and a large number of by-products due to chemical decomposition.
【０００３】また、インジゴを製造する種々の方法の中
で有望視されている方法として、キシレンオキシゲナー
ゼ又はナフタレンジオキシゲナ−ゼを含有する微生物を
酵素触媒として用い、インド−ルから製造する方法が知
られている（Ｂｕｒｔ Ｄ．Ｅｎｓｌｅｙ，Ｂａｒｒｙ
Ｊ．Ｒａｔｚｋｉｎ，Ｔｉｍｔｈｙ Ｄ．Ｏｓｓｌｕ
ｎｄ ａｎｄ Ｍａｒｙ Ｊ．Ｓｉｍｏｎ；Ｓｃｉｅｎ
ｃｅ，ｖｏｌ．２２２，Ｐ．１６７−１６９（１９８３
））。しかしながら、該酵素を工業的に使用するために
は、酵素活性を低下させることなく、また高収量で該酵
素含有微生物を得ることが必要であり、インジゴ生成能
を有する微生物の実際的な培養方法の開発が望まれてい
る。[0003] Among the various methods for producing indigo, one that is considered promising is a method for producing indigo from indole using microorganisms containing xylene oxygenase or naphthalene dioxygenase as an enzyme catalyst. Known (Burt D. Ensley, Barry
J. Ratzkin, Timthy D. Osslu
nd and Mary J. Simon; Science
ce, vol. 222, P. 167-169 (1983
)). However, in order to use the enzyme industrially, it is necessary to obtain a microorganism containing the enzyme in high yield without reducing the enzyme activity, and a practical method for culturing a microorganism capable of producing indigo is necessary. development is desired.
【０００４】また、従来、インジゴ生成能を有するシュ
ードモナス属に属する微生物を培養するときに、副原料
として、安息香酸又はその塩を用いることが知られてい
たが（Ｐ．Ａ．Ｗｉｌｌｉａｍｓ ａｎｄ Ｋ．Ｍｕｒ
ｒａｙ，Ｊ．Ｂａｃｔｅｒｉｏｌ．，１２０， ４１６
〜４２３（１９７４））、安息香酸やその塩は、微生物
のインジゴ生成能及び菌体収量に影響するという問題点
があった。[0004] Furthermore, it has conventionally been known to use benzoic acid or its salt as an auxiliary raw material when culturing microorganisms belonging to the genus Pseudomonas that have the ability to produce indigo (PA Williams and K. Mur
ray, J. Bacteriol. , 120, 416
423 (1974)), benzoic acid and its salts had a problem in that they affected the indigo production ability of microorganisms and the bacterial cell yield.
【０００５】[0005]
【発明が解決しようとする課題】本発明の目的は、イン
ジゴ生成菌のインジゴ生成能及び菌体収量を向上させる
ことができる培養方法を提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide a culture method capable of improving the indigo-producing ability and cell yield of indigo-producing bacteria.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、インジゴ
生成菌の工業的培養方法を確立するために、培養条件等
について鋭意検討した結果、安息香酸又はその塩の添加
濃度が０．４ （Ｗ／Ｖ）％を超えると、菌体の増殖及
び酵素の生成が阻害されることを見出し、本発明を完成
するに至った。[Means for Solving the Problems] In order to establish an industrial culture method for indigo-producing bacteria, the present inventors conducted intensive studies on culture conditions, etc., and found that the concentration of benzoic acid or its salt added was 0.4. (W/V)%, the growth of bacterial cells and production of enzymes are inhibited, and the present invention has been completed.
【０００７】すなわち、本発明は、培養中の安息香酸又
はその塩の培地濃度が０．４ （Ｗ／Ｖ）％を超えない
ように、安息香酸又はその塩を断続的又は連続的に添加
して培養することを特徴とする、インジゴ生成能を有す
るシュードモナス属に属する微生物の培養方法である。[0007] That is, in the present invention, benzoic acid or its salt is added intermittently or continuously so that the concentration of benzoic acid or its salt in the culture medium does not exceed 0.4 (W/V)%. This is a method for culturing a microorganism belonging to the genus Pseudomonas having the ability to produce indigo.
【０００８】以下、本発明を詳細に説明する。本発明の
培養方法は、インジゴ生成能を有するシュードモナス属
に属する微生物に利用することができるが、特にシュー
ドモナス（Ｐｓｅｕｄｏｍｏｎａｓ） ・ｓｐ．ＭＹ−
６菌株が好適に用いられる。本菌株の菌学的性質とその
分類学的性質は次の通りである。The present invention will be explained in detail below. The culture method of the present invention can be used for microorganisms belonging to the genus Pseudomonas that have the ability to produce indigo, and in particular, for Pseudomonas sp. MY-
6 strains are preferably used. The mycological properties and taxonomic properties of this strain are as follows.
【０００９】Ｉ．顕微鏡的性質
（ａ） 細胞の形及び大きさ：桿菌、１×２〜４μｍ（
ｂ） 多形性の有無：無し
（ｃ） 運動性：有り
（ｄ） 鞭毛の着生状態：極鞭毛
（ｅ） 胞子の有無：無し
（ｆ） グラム染色：陰性I. Microscopic properties (a) Cell shape and size: Bacillus, 1 x 2-4 μm (
b) Presence or absence of pleomorphism: Absent (c) Motility: Yes (d) Epiphytic status of flagella: Polar flagella (e) Presence or absence of spores: Absent (f) Gram staining: Negative
【００１０】ＩＩ．培養的性質
（ａ） 肉汁寒天培地における生育：有り（ｂ） 資化
可能な炭素源：グルコース、酢酸、コハク酸、フマル酸
、Ｌ−リンゴ酸、乳酸、クエン酸、グリセロール、Ｌ−
バリン、β−アラニン、ＤＬ−アルギニンII. Cultural properties (a) Growth on broth agar medium: Yes (b) Assimilable carbon sources: glucose, acetic acid, succinic acid, fumaric acid, L-malic acid, lactic acid, citric acid, glycerol, L-
Valine, β-alanine, DL-arginine
【００１１】
ＩＩＩ．生育条件
（ａ） 生育温度：１０〜４０℃
（ｂ） 生育ｐＨ：６〜８
（ｃ） 酸素要求性：好気性[0011]
III. Growth conditions (a) Growth temperature: 10-40°C (b) Growth pH: 6-8 (c) Oxygen requirement: aerobic
【００１２】ＩＶ．生理学的性質
（ａ） オキシダーゼ：陽性
（ｂ） カタラーゼ：陽性
（ｃ） ＤＮＡ中のグアニン、シトシン（ＧＣ）含量：
６１％
（ｄ） デンプンの加水分解：陰性
（ｅ） プロトカテキン酸の分解：陽性IV. Physiological properties (a) Oxidase: positive (b) Catalase: positive (c) Guanine and cytosine (GC) content in DNA:
61% (d) Hydrolysis of starch: Negative (e) Decomposition of protocatechinic acid: Positive
【００１３】以
上の諸性質を「バージーズ・マニュアル・オブ・システ
マティク・バクテリオロジー（Ｂｅｒｇｅｙ’ｓ Ｍａ
ｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒ
ｉｏｌｏｇｙ）」第２巻（１９８６年）より検索した。
その結果、本菌はシュードモナス（Ｐｓｅｕｄｏｍｏｎ
ｕｓ） 属に属する菌株であると同定されたが、種につ
いては炭素源の資化性その他の性質から合致しない点が
あり、新菌株と考えられた。従って本発明においてはシ
ュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ） ・ｓｐ．Ｍ
Ｙ−６と呼称することとする。なお本菌株は工業技術院
微生物工業技術研究所に微生物寄託番号微工研菌寄第１
１９６３号（ＦＥＲＭＰ−１１９６３）として寄託され
ている。The above properties are described in Bergey's Manual of Systematic Bacteriology.
natural of Systematic Bacter
Retrieved from "Iology)" Volume 2 (1986). As a result, this bacterium was found to be Pseudomonas (Pseudomonas).
Although it was identified as a strain belonging to the genus U.S., the species did not match in terms of its ability to assimilate carbon sources and other properties, so it was considered a new strain. Therefore, in the present invention, Pseudomonas sp. M
We will call it Y-6. This strain has been deposited with the Microbial Research Institute of the Agency of Industrial Science and Technology with the microorganism deposit number No. 1.
No. 1963 (FERMP-11963).
【００１４】炭素源としては、安息香酸又はその塩を用
い、培養中の濃度が、０．４ （Ｗ／Ｖ）％を超えない
ように、好ましくは０．３ （Ｗ／Ｖ）％を超えないよ
うに断続的又は連続的に培地に添加する。[0014] Benzoic acid or its salt is used as the carbon source, and the concentration during the culture does not exceed 0.4 (W/V)%, preferably exceeds 0.3 (W/V)%. Add to the culture medium intermittently or continuously to prevent
【００１５】窒素源としては、塩化アンモニウム、硫酸
アンモニウム、リン酸アンモニウム等のアンモニウム塩
；硝酸ナトリウム、硝酸カリウム、硝酸アンモニウム等
の硝酸塩；アンモニア等を用いることができる。As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate; nitrates such as sodium nitrate, potassium nitrate and ammonium nitrate; ammonia and the like can be used.
【００１６】無機物としては、リン酸カリウム、硝酸マ
グネシウム、鉄、マンガン、亜鉛等を用いることができ
る。また、必要に応じて、ビタミン、アミノ酸等の栄養
源を添加することができる。As the inorganic substance, potassium phosphate, magnesium nitrate, iron, manganese, zinc, etc. can be used. In addition, nutritional sources such as vitamins and amino acids can be added as necessary.
【００１７】培養温度は、２０℃〜５０℃、好ましくは
３０℃〜４０℃であり、培養中の培地のｐＨは６〜９、
好ましくは７〜８である。[0017] The culture temperature is 20°C to 50°C, preferably 30°C to 40°C, and the pH of the culture medium is 6 to 9.
Preferably it is 7-8.
【００１８】本発明により培養した菌体を本酵素反応に
利用する場合、該菌体はそのまま使用することができる
が、菌体の処理物、例えば該菌体を超音波処理等で破砕
した破砕物、又はその破砕物をさらに水等で抽出した抽
出物、或いは該抽出物をさらに硫安等で処理して酵素成
分を沈殿させた粗精製物の形で使用することもでき、さ
らに、該菌体又はこれら処理物を必要により固定化して
用いることもできる。When the bacterial cells cultured according to the present invention are used in the present enzyme reaction, the bacterial cells can be used as they are, but a processed product of the bacterial cells, for example, crushed cells obtained by crushing the bacterial cells by ultrasonication etc. It can also be used in the form of an extract obtained by further extracting the microorganism or its crushed product with water or the like, or a crudely purified product obtained by further treating the extract with ammonium sulfate or the like to precipitate the enzyme component. If necessary, the body or the treated product can be immobilized and used.
【００１９】該菌体又はその処理物の存在下でのインジ
ゴ生成の反応は、好気的に行い、従来の酵素反応と同様
に、インド−ル０．１〜２ｍＭを含む０．１Ｍ リン酸
緩衝液（ｐＨ６〜９）あるいは水溶液（ｐＨ６〜９）中
で、２０〜５０℃、好ましくは３０〜４０℃の温度で、
通常１０〜７２時間反応させる。反応は、撹拌しながら
行うのが好ましい。The reaction for producing indigo in the presence of the bacterial cells or their processed material is carried out aerobically, using 0.1M phosphoric acid containing 0.1 to 2mM indole as in the conventional enzyme reaction. in a buffer solution (pH 6 to 9) or an aqueous solution (pH 6 to 9) at a temperature of 20 to 50 °C, preferably 30 to 40 °C,
The reaction is usually carried out for 10 to 72 hours. The reaction is preferably carried out with stirring.
【００２０】反応に使用する微生物菌体又はその処理物
の添加量は、特に制限されるものではないが、一般に、
０．５〜１０％（ｗｔ／ｖｏｌ） が用いられる。反応
後、反応液からのインジゴの分離、精製は、それ自体該
知の方法、例えば、溶媒（クロロホルム）抽出等の方法
で行うことができる。[0020] The amount of microorganisms used in the reaction or their processed material is not particularly limited, but in general,
0.5-10% (wt/vol) is used. After the reaction, indigo can be separated and purified from the reaction solution by methods known per se, such as solvent (chloroform) extraction.
【００２１】[0021]
【実施例】（実施例１）（ＮＨ２ ）ＳＯ４ ：３ｇ，
ＫＨ２ ＰＯ４ ：０．５ｇ，Ｋ２ ＨＰＯ４ ：０．
５ｇ，ＭｇＳＯ４ ・７Ｈ２ Ｏ：０．５ｇ，ＣａＣｌ
２ ・２Ｈ２ Ｏ：１０ｍｇ，ＮａＣｌ：０．５ｇ，Ｆ
ｅＳＯ４ ・７Ｈ２ Ｏ：１０ｍｇ、酵母エキス１ｇ及
び蒸留水：１０００ｍｌ（ｐＨ７．０）の培地１００ｍ
ｌを５００ｍｌ容の三角フラスコに分注し、１２０℃、
１５分間滅菌処理したものに、インジゴ生成菌であるシ
ュードモナス・ＳＰ． ＭＹ−６菌株（ＦＥＲＭ Ｐ
−１１９６３）を植菌し、３０℃にて２４時間振盪培養
した。[Example] (Example 1) (NH2)SO4: 3g,
KH2 PO4: 0.5g, K2 HPO4: 0.
5g, MgSO4 ・7H2O: 0.5g, CaCl
2 ・2H2 O: 10 mg, NaCl: 0.5 g, F
eSO4 ・7H2 O: 10mg, yeast extract 1g and distilled water: 1000ml (pH 7.0) 100ml medium
1 into a 500 ml Erlenmeyer flask, and heated at 120°C.
After sterilization for 15 minutes, Pseudomonas sp., an indigo-producing bacterium, was added. MY-6 strain (FERM P
-11963) was inoculated and cultured with shaking at 30°C for 24 hours.
【００２２】また、上記と同様の滅菌培地１０００ｍｌ
を５０００ｍｌ容の三角フラスコに入れ、安息香酸ナト
リウムを表１に示す実験区の濃度になるように添加した
後、上記振盪培養液２０ｍｌを接種し、これを３０℃に
て２４時間振盪培養した。得られた培養液の１００ｍｌ
を遠心分離（８０００ｒｐｍ ，１５分間、４℃）して
集菌した。その集菌菌体の湿菌体重量を測定し、安息香
酸ナトリウムの濃度が０．３ （Ｗ／Ｖ）％のときの湿
菌体重量を１００として換算した各安息香酸ナトリウム
の湿菌体重量の相対値を菌体収量（％）として求めた。
結果を表１に示す。[0022] Also, 1000 ml of the same sterilized medium as above.
was placed in a 5,000 ml Erlenmeyer flask, sodium benzoate was added to the concentration of the experimental group shown in Table 1, and then 20 ml of the above shaken culture solution was inoculated and cultured with shaking at 30° C. for 24 hours. 100 ml of the obtained culture solution
The cells were collected by centrifugation (8000 rpm, 15 minutes, 4°C). The wet bacterial weight of the collected bacteria was measured, and the wet bacterial weight of each sodium benzoate was calculated based on the wet bacterial weight when the concentration of sodium benzoate was 0.3 (W/V)% as 100. The relative value of was determined as bacterial cell yield (%). The results are shown in Table 1.
【００２３】上記の方法で調製した菌体を酵素源とし、
反応液（１００ｍＭリン酸緩衝液 ｐＨ７．０）１００
ｍｌに懸濁後、インド−ル１ｍＭを添加し、振盪しなが
ら３０℃で２４時間反応させた。生成したインジゴ量を
、常法［Ｈ． ＫＥＩＬ， Ｃ．Ｍ． ＳＡＩＮＴ ａ
ｎｄ Ｐ．Ａ． ＷＩＬＬＩＡＭＳ， Ｊｏｕｒｎａｌ
ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ， １６９，Ｎｏ．
２，Ｐ．７６４−７７０（１９８７） ］に従い定量し
、安息香酸ナトリウムの濃度が０．３ （Ｗ／Ｖ）％の
ときのインジゴ量を１００として換算した各安息香酸ナ
トリウムのインジゴ量の相対値をインジゴ生成量（％）
として求めた。結果を表１に示す。[0023] Using the bacterial cells prepared by the above method as an enzyme source,
Reaction solution (100mM phosphate buffer pH 7.0) 100
ml, 1 mM of indole was added, and the mixture was reacted at 30° C. for 24 hours with shaking. The amount of indigo produced was determined by a conventional method [H. KEIL, C. M. SAINT a
nd P. A. WILLIAMS, Journal
of Bacteriology, 169, No.
2, P. 764-770 (1987)], and the indigo amount of each sodium benzoate was calculated based on the indigo amount when the concentration of sodium benzoate is 0.3 (W/V)% as 100. The indigo production amount is the relative value of the indigo amount of each sodium benzoate. (%)
I asked for it as. The results are shown in Table 1.
【００２４】[0024]
【表１】[Table 1]
【００２５】[0025]
【発明の効果】本発明の方法によれば、インジゴ生成菌
のインジゴ生成能及び菌体収量を向上させることができ
るので、高収率でインジゴを製造することができる。According to the method of the present invention, the indigo-producing ability of indigo-producing bacteria and the bacterial cell yield can be improved, so that indigo can be produced at a high yield.
Claims (1)
度が０．４ （Ｗ／Ｖ）％を超えないように、安息香酸
又はその塩を断続的又は連続的に添加して培養すること
を特徴とする、インジゴ生成能を有するシュードモナス
属に属する微生物の培養方法。Claim 1: Culture by adding benzoic acid or its salt intermittently or continuously so that the concentration of benzoic acid or its salt in the culture medium does not exceed 0.4 (W/V)%. A method for culturing a microorganism belonging to the genus Pseudomonas having indigo-producing ability, characterized by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11077791A JPH04320680A (en) | 1991-04-17 | 1991-04-17 | Culture of bacterium belonging to genus pseudomonas, capable of forming indigo |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11077791A JPH04320680A (en) | 1991-04-17 | 1991-04-17 | Culture of bacterium belonging to genus pseudomonas, capable of forming indigo |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04320680A true JPH04320680A (en) | 1992-11-11 |
Family
ID=14544343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11077791A Pending JPH04320680A (en) | 1991-04-17 | 1991-04-17 | Culture of bacterium belonging to genus pseudomonas, capable of forming indigo |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04320680A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060218A (en) * | 2011-10-18 | 2013-04-24 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
-
1991
- 1991-04-17 JP JP11077791A patent/JPH04320680A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060218A (en) * | 2011-10-18 | 2013-04-24 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
| CN103060218B (en) * | 2011-10-18 | 2014-04-23 | 大连理工大学 | Phenol-degrading bacteria and method for preparing indigo by conversing indole |
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