WO2010079763A1 - Method for producing indolylalkyl succinic acid amide compound - Google Patents

Method for producing indolylalkyl succinic acid amide compound Download PDF

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WO2010079763A1
WO2010079763A1 PCT/JP2010/000089 JP2010000089W WO2010079763A1 WO 2010079763 A1 WO2010079763 A1 WO 2010079763A1 JP 2010000089 W JP2010000089 W JP 2010000089W WO 2010079763 A1 WO2010079763 A1 WO 2010079763A1
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bacillus
strain
acid
iesa
mass
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Japanese (ja)
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亨 北村
洋 副島
寛司 上西
民二 杉山
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雪印種苗株式会社
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    • C—CHEMISTRY; METALLURGY
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/11—Bacillus megaterium

Definitions

  • the present invention relates to a process for producing an indolylalkyl succinamide compound useful as a plant growth regulator using a microorganism.
  • This succinic acid amide compound can be obtained by chemical synthesis as described in Patent Documents 1 and 2, and phenethyl succinic acid amide is known to be produced by microorganisms belonging to the genus Bacillus (non-patent). Reference 1).
  • an object of the present invention is to provide a novel method for producing an indolylalkylsuccinamide compound useful as a plant growth promoter by a microorganism.
  • the present inventor searched for a new microorganism capable of producing indolylalkylsuccinic acid amide, and microorganisms belonging to the genus Bacillus efficiently produced indolylalkylsuccinic acid amide compounds from tryptamine, tryptophan and serotonin hydrochloride.
  • the present invention has been completed.
  • the present invention has the general formula (1)
  • R 1 and R 2 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group or an alkyl group, R 3 represents a hydrogen atom or a carboxyl group, and n represents a number of 0 to 3)
  • a microorganism belonging to the genus Bacillus having the ability to synthesize indolylalkylsuccinic acid amide is cultured in a medium containing the indolylalkylamines or salts thereof represented by the general formula (2)
  • an indolylalkylsuccinamide compound or a salt thereof useful as a plant growth regulator can be efficiently obtained by culturing a microorganism belonging to the genus Bacillus.
  • mass% means (wt / vol)%
  • volume% means (vol / vol)%
  • Microorganisms used in the method for producing an indolylalkylsuccinamide compound of the present invention include Bacillus thuringiensis , Bacillus cereus , Bacillus subtilis , Bacillus acidiceler. ), Bacillus Ashidikora (Bacillus acidicola), Bacillus Aeoriusu (Bacillus aeolius), Bacillus Aeriusu (Bacillus aerius), Bacillus Aerofirusu (Bacillus aerophilus), Bacillus Agaradoherensu (Bacillus agaradhaerens), Bacillus Akibai (Bacillus akibai) , Bacillus alkaline Filth (Bacillus alcalophilus), Bacillus Arugikora (Bacillus algicola), Bacillus alkaline diazo Toro Ficus (Bacillus alkalidiazotrophicus), Bacillus Arukariteruri Scan (Bacillus alkalitelluri
  • Bacillus badius Bacillus badius (Bacillus badius), Bacillus Babarikasu (Bacillus barbaricus), Bacillus Batabienshisu (Bacillus bataviensis), Bacillus Benzoboransu (Bacillus benzoevorans), Bacillus Bogorienshisu (Bacillus bogoriensis), Bacillus Boronifirusu (Bacillus boroniphilus), Bacillus Butanoriboransu (Bacillus butanolivorans), Bacillus Kabonifirusu (Bacillus carboniphilus), Bacillus Sesenbenshisu (Bacillus cecembensis), Bacillus cell Russia utility dregs (Bacillus cellulosilyticus), Bacillus Chaganorenshisu (Bacillus chagannorensis), Bacillus CB (Bacillus cibi) , Bacillus circulans , Bacillus
  • Bacillus Horikoshi Bacillus horikoshii
  • Bacillus Horuti Bacillus Horuti (Bacillus horti)
  • Bacillus Fumi Bacillus humi
  • Bacillus file Jin Po-ene-cis Bacillus hwajinpoensis
  • Bacillus Idorienshisu Bacillus idriensis
  • Bacillus Insoritasu Bacillus insolitus
  • Bacillus Isaberie Bacillus isabeliae
  • Bacillus Chokkari Bacillus jeotgali
  • Bacillus Korienshisu Bacillus koreensis
  • Bacillus krulwichiae Bacillus lehensis
  • Bacillus lentus Bacillus licheniformis
  • Bacillus pseudalcaliphilus Bacillus pseudofirmus , Bacillus pseudomycoides , Bacillus psychrodurans , Bacillus physaccharolyticus , Bacillus Fi black tolerance (Bacillus psychrotolerans), Bacillus pumilus (Bacillus pumilus), Bacillus Pikunasu (Bacillus pycnus), Bacillus King Dao Nene cis (Bacillus qingdaonensis), Bacillus Rurisu (Bacillus ruris), Bacillus Safenshisu ( Bacillus safensis), Bacillus Sarariusu (Bacillus salarius), Bacillus Sarifirusu (Bacillus saliphilus), Bacillus Sri Jerry (Bacillus schlegelii), Bacillus Serenatasena Office (Bacillus selenatarsenatis), Bacillus Serenity TOLED
  • Preferred examples of the above include Bacillus thuringiensis JCM20386, B-2 and B-3 (postal code 292-0818 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-689), B-9, B-10, B-18 (NITE ABP-858), B-21, B -22, B-24 (NITE AP-857), B-26, B-33, B-36, B-59; Bacillus cereus B-5, B -6 (NITE AP-690), B-7, B-8, B-11, B-12, B-14, B-15, B-16 Strain (NITE AP-856); Bacillus megaterium B-32 (NITE ABP-859) strain Bacillus subtilis JCM1465 strain and the like.
  • Bacillus thuringiensis JCM20386 strain, B-3 strain, B-18 strain, B-24 strain; Cereus B-6 strain, B-15 strain, B-16 strain; Bacillus subtilis JCM1465 strain is particularly preferable.
  • the deposit date is NITE AP-689 shares and NITE AP-690 shares are December 5, 2008, and the receipt numbers NITE AP-856, AP-857, ABP-858, ABP- 859 is December 25, 2009.
  • Bacillus thuringiensis JCM20386 strain and Bacillus subtilis JCM1465 strain are strains that can be purchased from RIKEN. Other strains were isolated from soil.
  • the medium contains an indolylalkylamine or a salt thereof represented by the general formula (1) Let me.
  • R 1 and R 2 include a hydrogen atom, a halogen atom, a hydroxyl group, a C 1-6 alkyl group, and a C 1-6 alkoxy group.
  • examples of the C 1-6 alkyl group include a methyl group, an ethyl group, an isopropyl group, and a t-butyl group.
  • examples of the C 1-6 alkoxy group include a methoxy group, an ethoxy group, and an isopropyloxy group.
  • a halogen atom a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom are mentioned.
  • n represents a number from 0 to 3, with 0 to 2, particularly 1 being preferred.
  • R 1 and R 2 are particularly preferably a hydrogen atom or a hydroxyl group, and n is particularly preferably 1.
  • examples of the salt of indolylalkylamines (1) include hydrochloride, nitrate, sulfate and the like, and hydrochloride is preferable.
  • the concentration of the indolylalkylamines (1) or a salt thereof in the medium is 0.001 to 10% by mass, more preferably 0.001 to 5% by mass, particularly 0 in terms of productivity of the indolylalkylsuccinic acid amide compound. 0.01 to 5% by mass is preferable.
  • the indolylalkylamines (1) can be added to the reaction system all at once or continuously.
  • manganese ions (Mn 2+ ) By containing manganese ions (Mn 2+ ) in the medium, an indolylalkyl succinamide compound can be produced more efficiently.
  • any of manganese sulfate, manganese hydrochloride, manganese carbonate, EDTA manganese, and the like can be used, and one or more of these can be used, and manganese sulfate is particularly preferable.
  • the content in the medium is preferably 1 to 2000 ppm, particularly 2 to 1000 ppm as manganese ions (Mn 2+ ).
  • Manganese ions (Mn 2+ ) can be added to the reaction system all at once or continuously.
  • an indolyl alkyl succinamide compound can be produced more efficiently by containing an organic acid or a salt thereof in the medium.
  • the organic acid include malic acid, fumaric acid, succinic acid, citric acid, isocitric acid, ⁇ -ketoglutaric acid, oxaloacetic acid, and the like, and one or more of these can be used.
  • malic acid, fumaric acid, and succinic acid are preferable, and L-malic acid, D-malic acid, and fumaric acid are more preferable in terms of production efficiency of indolylalkyl succinic acid amide compounds.
  • the concentration of these organic acids in the medium is preferably 0.1 to 10% by mass, more preferably 0.2 to 1% by mass.
  • the organic acid or a salt thereof can be added to the reaction system all at once or continuously.
  • the microorganism is cultured under the conditions under which normal microorganisms of the genus Bacillus grow, except that indolylalkylamines (1) or a salt thereof and, if necessary, other compounds described above are added to the medium. Just do it. It is preferable to add a carbon source, a nitrogen source, a vitamin source, a mineral source and the like necessary for the growth of Bacillus microorganisms to the medium.
  • a carbon source glucose, starch syrup, soluble starch and the like are used.
  • the nitrogen source tryptone, soyton, yeast extract, peptone, casein, casein digest, meat extract, corn steep liquor, amino acid solution, soybean cake, ammonium sulfate, ammonium chloride, etc. can be used, but casein with a high tryptophan content. Needless to say, casein digest, yeast extract, corn steep liquor and the like are preferable.
  • the mineral source magnesium salt, potassium salt, phosphate, iron salt and the like can be used.
  • whey such as milk; cheese whey, soybean whey, etc. can be used.
  • a shaking culture and a stirring culture may be performed using a liquid medium.
  • the culture conditions are aerobic conditions, preferably 8 to 45 ° C. and pH 3.2 to 9.5.
  • the culture time may be 24 to 720 hours.
  • the indolylalkyl succinamide compound (2) or a salt thereof may be collected by a conventional method.
  • This culture solution can also be used as a plant growth regulator or fertilizer as it is.
  • the indolylalkylsuccinic acid amide compound (2) or a salt thereof can be partially purified or purified and isolated by ion exchange resin, porous synthetic adsorbent, solvent extraction, or the like.
  • a partially purified product using an ion exchange resin is prepared by adjusting the pH of the culture medium to a neutral region (pH 5 to 8), adsorbing it on a strong basic ion exchange resin or a weak basic ion exchange resin, and then containing an acid. It can be obtained by elution with an aqueous alcohol solution. At this time, it is preferable to replace the ion exchange resin before adsorption with a formic acid type or an acetic acid type.
  • the acid concentration of the solution is preferably 0.01 to 4N, and more preferably 1 to 3N.
  • the acid include formic acid, acetic acid, hydrochloric acid, sulfuric acid and the like.
  • the alcohol concentration of the solution is preferably 0 to 80% by volume, more preferably 0 to 30% by volume. Examples of the alcohol include methanol, ethanol, butanol, isopropanol and the like.
  • a polystyrene synthetic adsorbent after adjusting the pH of the culture solution to an acidic to neutral range (pH 1 to 8), a polystyrene synthetic adsorbent, a styrene-divinylbenzene adsorbent, or a methacrylic adsorbent is used.
  • adsorbing to the system synthetic adsorbent it can be obtained by elution with an aqueous alcohol solution or an aqueous ketone solution.
  • the concentration of alcohol or ketone in the solution is preferably 5 to 99% by volume, more preferably 10 to 30% by volume.
  • the alcohol include methanol, ethanol, butanol, isopropanol and the like.
  • the ketone include acetone.
  • This culture solution may contain phenylacetic acid or indoleacetic acid having a plant growth regulating action (Kim et al. 2004. Current Microbiology 48, 312-317 and Willkinson et al. 1994. Plant and Soil, 159, 291). -295 pages).
  • the mixture may be used as it is, or each of them may be collected and used.
  • N- (phenethyl) succinamide may be contained, but in this case as well, the mixture may be used as it is depending on the purpose, or each of them may be used separately. .
  • plants can generally biosynthesize tryptophan, and that many plants can also biosynthesize tryptamine (Facchini et al. 2000. Phytochemistry, Vol. 54, pages 121-138). And it is known that some of them are secreted from the plant surface, especially from the root surface (Kamilova et al. 200. Molecular Plant-Microbe Interaction, Vol. 19, pages 250-256). For this reason, if these microbial strains are applied to or inoculated with these plants, their seeds, water for plant cultivation or soil, indolylalkyl succinic acid amide is biosynthesized in the plant body or on the plant body surface. Can be controlled.
  • an inoculation source a culture solution containing bacterial cells and / or spores obtained by culturing the strain under conditions in which normal Bacillus microorganisms are grown, centrifugation, membrane separation, etc. What concentrated the microbial cell and / or spore, or what wash
  • These inoculation sources can be used as they are, or dried or lyophilized products thereof. Further, it can be used by being supported on an inorganic substance carrier such as bentonite, zeolite, or clay, or an organic substance carrier such as peat moss or activated carbon.
  • the inoculation target can be used for whole plants, water and soil, but when utilizing the high rooting promoting activity of the compound, seeds, roots, cutting cross sections, etc. are preferable.
  • the inoculation concentration may be inoculated after the number of bacteria is 1 ⁇ 10 4 to 1 ⁇ 10 11 per gram of inoculation source.
  • R 4 represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group or an alkyl group, and m represents a number of 0 to 3
  • the compound represented by the general formula (3) or a salt thereof can be used as a rooting promoter or a plant growth regulator, and for producing a rooting promoter or a plant growth regulator.
  • promoting rooting means promoting rooting of plants such as azuki bean and tomato
  • controlling plant growth means adjusting the growth of the plant.
  • R 4 and m are the same as R 1 and n, respectively, in terms of rooting promoting action.
  • bubutanoic acid is particularly preferable from the viewpoint of rooting promoting action.
  • Example 1 Tryptic Soy Broth (manufactured by Becton Dickinson & Company) with 0%, 0.01%, 0.03%, 0.1%, or 0.3% by weight tryptamine added to a 500 mL Erlenmeyer flask with baffle
  • the following TSB were prepared in an amount of 2 x 300 mL, autoclaved, and inoculated with Bacillus thuringiensis JCM20386 strain that had been pre-cultured with TSB in advance.
  • the cells were cultured with shaking at a temperature of 27 ° C. and a rotation speed of 80 rpm for 10 days.
  • the culture temperature was 27 ° C., and shaking culture was performed at a rotation speed of 80 rpm for 10 days.
  • the obtained culture broth was roughly purified as follows.
  • the culture solution was centrifuged at 6,500 rpm for 30 minutes, and the supernatant was adjusted to pH 3.0 with hydrochloric acid.
  • styrene-divinylbenzene synthetic adsorption resin Diaion HP-20 was washed with methanol, then packed in a column (inner diameter 20 mm ⁇ length 300 mm), and adjusted by passing pH 3.0 acetic acid solution through. By flowing the supernatant liquid through this column, N- [2- (3-indolyl) ethyl] succinamide (hereinafter, IESA) was adsorbed.
  • IESA N- [2- (3-indolyl) ethyl] succinamide
  • the column was washed with 500 mL of 10% by volume isopropanol, and IESA was eluted with 500 mL of 30% by volume isopropanol.
  • the eluate was concentrated to about 100 mL with an evaporator, adjusted to pH 8.0 using an aqueous sodium hydroxide solution, extracted three times with ethyl acetate, and the ethyl acetate layer was discarded.
  • the remaining aqueous phase was adjusted to pH 2.5 with hydrochloric acid and extracted three times with ethyl acetate.
  • the obtained ethyl acetate layer was dehydrated using anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure.
  • the residue was dissolved in a small amount of 1N aqueous acetic acid, separately washed with methanol, and passed through a SepPak C18 cartridge (manufactured by Waters) prepared by passing through 1N aqueous acetic acid to adsorb IESA.
  • the cartridge was eluted with 20 mL of 60% by volume methanol, and the eluate was evaporated to dryness under reduced pressure.
  • This sample was purified using HPLC (column, YMC-Pack Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol by volume containing 1% acetic acid; flow rate, 3.0 mL / min) The fraction corresponding to the IESA retention time (retention time 8.5 to 11.5 minutes) was collected. The fraction collected was purified using HPLC (column, ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the IESA retention time (retention time 11 to 13 minutes) was collected.
  • HPLC columnumn, YMC-Pack Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol by volume containing 1% acetic acid; flow rate, 3.0 mL / min
  • this strain produced IESA in the culture medium even when cultured with TSB alone. This is considered to be derived from the case where about 1% by mass of tryptophan derived from casein added as a medium raw material in TSB is contained. Furthermore, it was also proved that IESA production was dramatically improved in the area where tryptamine was added.
  • the fraction corresponding to the IESA retention time (retention time 13.5 to 15.5 minutes) was collected, concentrated for 5 samples into one sample, and further HPLC (column, XTerraRP inner diameter 10 mm ⁇ long). 250 mm (manufactured by Waters); mobile phase, 25% acetonitrile by volume containing 1% acetic acid; flow rate, 3.0 mL / min), and fraction corresponding to IESA retention time (retention time 15-19 minutes) was single Sorted as a peak. The obtained fraction was concentrated under reduced pressure and then dried under reduced pressure in a desiccator in the presence of diphosphorus pentoxide. As a result, 5.8 mg of IESA crystals were obtained.
  • Example 2 Two 500 mL Nutrient Broth (manufactured by Becton Dickinson & Company, NB) with 1% by mass tryptophan added to a 500 mL Erlenmeyer flask with baffle was prepared, and after Bacillus thuringiensis B precultured with NB after autoclaving. -3 strains (from Betsukai-cho soil in Hokkaido, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-689) and B. Cereus B-6 strain (derived from Betsukai-cho, Hokkaido, receipt number NITE AP-690) was inoculated and cultured in the same manner as in Example 1.
  • B-6 strain gene identification was performed using the 16s rDNA sequence according to the following procedure. Bacterial cells were collected from the culture solution of B6 strain aerobically cultured at 37 ° C. for 2 days with NB by centrifugation, and DNA was extracted with PrepMan Ultra (Applied Biosystems).
  • PCR was performed at 94 ° C for 4 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 5 minutes.
  • the amplified product was purified with MinElute PCR purification kit (QIAGEN), and the partial sequence analysis of the amplified fragment was requested to SIGMA Genosys. About 700 bp analyzed, FASTA search was performed on the DDBJ (DNA Data Bank of Japan) homepage (http://fasta.ddbj.nig.ac.jp/top-j.html), and the homology was highest. Identified as a bacterial species.
  • the culture solution was roughly purified in the same manner as in Example 1.
  • the crudely purified sample was purified using HPLC (column, YMC Pack C8 inner diameter 20 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 6.0 mL / min).
  • the fraction corresponding to the IESA retention time was collected.
  • Further fractions were collected using HPLC (column, YMC Pack C8 inner diameter 20 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 30% acetonitrile containing 1% acetic acid; flow rate, 6.0 mL / min).
  • IESA retention time Purified and fractions corresponding to IESA retention time (retention time 28 to 34 minutes) were collected. Further, the fractions used were HPLC (column, YMC-Pack Phe, inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the IESA retention time (retention time 9 to 11 minutes) was collected. Further, the fractions were collected using HPLC (column, ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min).
  • fraction collected was HPLC (column, Puresil C18, inner diameter 4.6 mm ⁇ length 250 mm (manufactured by Waters); column temperature, 40 ° C .; mobile phase, 25% acetonitrile containing 1% by volume acetic acid; 8 mL / min).
  • HPLC column temperature, 40 ° C .
  • mobile phase 25% acetonitrile containing 1% by volume acetic acid; 8 mL / min.
  • a peak was observed at 12.4 minutes, which almost coincided with chemically synthesized IESA.
  • an absorption maximum was observed at 281 nm, which was consistent with chemically synthesized IESA.
  • Example 3 Culture, rough purification, and analysis were performed in the same manner as in Example 2 except that the medium was TSB to which 3% by volume of corn steep liquor (manufactured by Wako Pure Chemical Industries, Ltd.) was added and the strain was B-3. As a result, 0.102 mg / L IESA was detected.
  • Example 4 The culture medium is NB, NB with 10 mM tryptamine added, NB with 10 mM tryptamine added with 3 mM phenethylamine, NB with 10 mM tryptamine and 10 mM phenethylamine added, and otherwise with Example 1. Similarly, culture and crude purification were performed, respectively.
  • the crudely purified sample was obtained using HPLC (column, YMC-Pack Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min).
  • HPLC column, YMC-Pack Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min).
  • fractions corresponding to retention times of IESA and N- (phenethyl) succinamide (hereinafter referred to as PESA) retention times of 8.5 to 11 minutes
  • the fraction collected was purified using HPLC (column, ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min).
  • the fraction corresponding to the retention time of IESA and PESA was collected.
  • Further fractions were analyzed by HPLC (column, Polar RP inner diameter 10 mm ⁇ length 250 mm (Phenomenex); mobile phase, 30% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min).
  • An IESA peak was observed at 14.0 minutes, which almost coincided with chemically synthesized IESA.
  • Example 5 Prepare NB containing 0.1% by mass of tryptamine in a 500 mL Erlenmeyer flask with baffle, and inoculate with Bacillus subtilis strain JCM1465 pre-cultured with NB after autoclaving and culture as in Example 1. Crude purification was performed. The crudely purified sample was purified and analyzed by HPLC in the same manner as in Example 4. As a result, 0.032 mg / L IESA was detected. Therefore, B. be added tryptamine appropriate concentrations in the medium It became clear that IESA could be produced using subtilis .
  • Example 6 Medium containing 1% by mass of tryptophan in NB, 1% by mass of tryptophan in NB, and 0.3% by mass of manganese sulfate pentahydrate (MnSO 4 .5H 2 O) (684 ppm as manganese ion concentration) Then, it was adjusted to pH 6.8 with an aqueous sodium hydroxide solution, and cultured, roughly purified and analyzed in the same manner as in Example 5 except that the strain was changed to the B-3 strain. As a result, IESA of 0.643 mg / L was detected in the manganese sulfate-free group and 3.704 mg / L in the manganese sulfate-added group. From this, it was confirmed that the addition of manganese ions increased IESA production by more than 5 times.
  • MnSO 4 .5H 2 O manganese sulfate pentahydrate
  • Example 7 The basic medium is 0.5% by mass of sodium chloride (NaCl), 0.25% by mass of dipotassium hydrogen phosphate (K 2 HPO 4 ), 0.25% by mass of glucose (glucose), manganese sulfate pentahydrate (0.3%). (Mass%, manganese ion concentration 684 ppm).
  • Yeast extract Yeast extracts, manufactured by Becton Dickinson & Company
  • Tryptone Tryptone, manufactured by Becton Dickinson & Company
  • Soyton Soytone, Becton -Dickinson & Company, Inc.
  • Example 3 except that 3% by mass, (4) 3% by mass of soyton and 1% by mass of glutamic acid were added to adjust the pH to 7.2, and the strain was B-3.
  • culture, crude purification, and analysis were performed. The results are shown in Table 4. From this result, it was confirmed that IESA can be produced from a wide range of nitrogen sources such as yeast extract, tryptone, and soyton by adding manganese ions to the medium.
  • Example 8 (1) Unadjusted milk (manufactured by Nippon Milk Community Co., Ltd.) itself, (2) 0.3% by mass of L-malic acid added to the milk, (3) Manganese sulfate pentahydrate added in an amount of 0.3% by mass (manganese ion concentration of 684 ppm), (4) 0.3% by mass of L-malic acid and manganese sulfate pentahydrate in the above milk Each was added with 0.3% by mass of the product, adjusted to pH 7.2, used as a medium, and cultured, roughly purified and analyzed in the same manner as in Example 5 except that the strain was B-3 strain. The results are shown in Table 5. From this result, it became clear that IESA can be produced using B-3 strain even when milk itself is used as a medium. It was also found that the production amount was further improved by adding manganese ions and malic acid.
  • Example 9 L-malic acid (L-malic acid) was added to whey UF membrane permeate (manufactured by Snow Brand Milk Products Co., Ltd., Nakashibetsu Factory), which is a permeate when protein is recovered from cheese whey using a UF membrane (Ultrafiltration Membrane).
  • IESA can be produced when whey UF membrane permeate itself is used as a medium and manganese ions and L-malic acid are added.
  • whey such as whey powder
  • IESA can be produced by the B-3 strain.
  • manganese ions and L-malic acid are added, the production can be dramatically improved. Became clear.
  • Example 10 Protein concentrated whey powder WPC34C (concentrated protein in whey, spray-dried; manufactured by Snow Brand Milk Products Co., Ltd.) 3% by mass, sodium chloride (NaCl) 0.5% by mass, dipotassium hydrogen phosphate (K 2 HPO) 4 ) Whey protein basal medium was prepared by dissolving 0.25% by mass and 0.25% by mass of glucose in distilled water.
  • IESA can be produced by using the B-3 strain even when the whey protein itself is used as a medium. Further, the production amount was further improved by adding manganese ions and organic acids, and it was found that D-malic acid and fumaric acid are effective as organic acids in addition to L-malic acid.
  • Example 11 (1) Semi-desalted whey powder FC10 (manufactured by Snow Brand Milk Products Co., Ltd.) 12% by mass aqueous solution, (2) Manganese sulfate pentahydrate is added to the 12% by mass semi-desalted whey powder aqueous solution in an amount of 0.3% by mass. (Manganese ion concentration 684 ppm) added, (3) manganese sulfate pentahydrate 0.3% by mass and D-malic acid 0.3% by mass added to the above aqueous solution, (4) sulfuric acid added to the above aqueous solution Manganese pentahydrate 0.3% by mass and L-malic acid 0.3% by mass added.
  • Example 12 Semi-desalted whey powder FC10 (manufactured by Snow Brand Milk Products Co., Ltd.) A medium adjusted to pH 7.2 by adding 0.3% by mass of manganese sulfate pentahydrate (684 ppm as manganese concentration) to a 12% by mass aqueous solution (implemented) 300 mL of Example 11 (2)) was prepared, put into a 500 mL Erlenmeyer flask with a baffle, and sterilized by autoclave.
  • Bacillus thuringiensis B-2 strain (derived from Hokkaido Betsukai-cho soil), B-9 strain (derived from Naganuma-cho soil in Hokkaido), B-10 strain (derived from Betsukai-cho soil in Hokkaido) B-18 strain (from Hokkaido Yuni-cho soil, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE ABP-858), B-21 strain (from Hokkaido Yuni-cho soil), B-22 Shares (derived from Naganuma Town, Hokkaido), B-24 shares (derived from Betsukai Town, Hokkaido, (Japan) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-857), B-26 shares (Shibetsu Town, Hokkaido) Soil origin), B-33 strain (derived from Naganuma-cho soil in Hokkaido), B-36 strain (derived from soil in Shibetsu-cho, Hokkaido), B-59 strain (derived from soil in
  • B-7 strain (Derived from Naganuma Town, Hokkaido), B-1 Ltd. (Hokkaido Naganuma-cho, derived from the soil), the B-16 share (Hokkaido Naganuma-cho, soil-derived, (Germany) Technology and Evaluation board mechanism, Patent Microorganisms Depositary receipt number NITE AP-856);
  • B. megaterium B-32 strain (Hokkaido Yuni-cho soil origin, (Germany) product evaluation technology substrate mechanism patent microorganism deposit center receipt number NITE ABP-859) was inoculated and cultured in the same manner as in Example 1. The above strains were identified by the same method as in Example 2.
  • the culture solution was roughly purified in the same manner as in Example 1.
  • the crudely purified sample was purified using HPLC (column, YMC ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min).
  • the fraction corresponding to the IESA retention time was collected.
  • the fraction collected was HPLC (column, Polar RP inner diameter 4.6 mm ⁇ length 250 mm (manufactured by Phenomenex)); column temperature, 40 ° C .; mobile phase, 25% acetonitrile by volume containing 1% by volume acetic acid; 8 mL / min).
  • a peak was observed at 14.4 minutes, which almost coincided with chemically synthesized IESA.
  • the IESA concentration was calculated in the same manner as in Example 1 using the peak area measured at 280 nm. The results are shown in Table 9. From this result, it was confirmed that IESA can be produced using B. thuringiensis , B. cereus , B. megaterium by using whey supplemented with manganese as a medium. In particular, the production capacity of B. thuringiensis B-18 strain (NITE ABP-858) was found to be extremely high.
  • N- [2- (5-hydroxyindol-3-yl) ethyl] succinic acid amide (Bufobutanoic acid, hereinafter referred to as BBA) was chemically synthesized as follows. Add 30 mL of acetone to 1.55 g of succinic anhydride and stir at room temperature with 3 g of 5-hydroxytryptamine hydrochloride (serotonin hydrochloride, Sigma-Aldrich Japan Co., Ltd.) and 1.97 mL of triethylamine in 50 mL of acetone.
  • the synthesized product was obtained using HPLC (column, YMC-Pack C8 inner diameter 20 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 6.0 mL / min). The eluate was purified and the retention time was 12-14 minutes. The fractions collected were dried under reduced pressure and dried under reduced pressure in a desiccator in the presence of diphosphorus pentoxide to obtain 1.1 g of BBA.
  • Production by culture was carried out as follows. Add 0.1% by mass of 5-hydroxytryptamine hydrochloride to NB and adjust to pH 6.8, then dispense 300 mL each into two 500 mL baffled Erlenmeyer flasks, sterilize by autoclave, and use NB in advance.
  • the pre-cultured Bacillus thuringiensis B-3 strain and B. cereus B-6 strain (from Betsukai-cho, Hokkaido, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center Accession Number NITE AP-690) were inoculated.
  • the culture temperature was 37 ° C., and shaking culture was performed at a rotation speed of 80 rpm for 10 days.
  • the obtained culture broth was centrifuged in the same manner as in Example 1 and then adsorbed onto a Diaion HP-20 column. This column was eluted with 500 mL of 10% by volume isopropanol, and the eluate was subjected to solvent extraction in the same manner as in Example 1.
  • the obtained sample was dissolved in a small amount of 20% by volume methanol, separately washed with methanol, passed through a SepPak tC18 cartridge (Waters) prepared by passing 20% by volume methanol, and further 20 mL 20% by volume methanol. Purified by eluting with
  • the crudely purified sample was purified using HPLC (column, YMC Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 30% methanol containing 1% acetic acid; flow rate, 3.0 mL / min)
  • HPLC columnumn, YMC Phe inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 30% methanol containing 1% acetic acid; flow rate, 3.0 mL / min
  • the fraction corresponding to the BBA retention time was collected.
  • fractions collected were analyzed by HPLC (column, ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 20% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min). .
  • HPLC column, ODS-A inner diameter 10 mm ⁇ length 250 mm (manufactured by YMC); mobile phase, 20% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min).
  • a peak was observed at 9.5 minutes, which almost coincided with chemically synthesized BBA.
  • the absorption maximum of the peak coincided with 279 nm and chemically synthesized BBA, and the absorption spectrum of 235 to 400 nm almost coincided with chemically synthesized BBA.
  • BBA has root-promoting activity
  • a chemically synthesized BBA aqueous solution was prepared and adjusted to pH 7 with hydrochloric acid, and azuki root-promoting assay (Itagaki et al. 2003. Biological activities and structure-activity).
  • Azuki bean slices were immersed in the test solution for 72 hours, and the number of adventitious roots generated after 7 days was counted. The number of iterations was 5. The results are shown in Table 10. In the BBA-treated group, a marked increase in the number of roots was observed depending on the concentration. From this, it was confirmed that said BBA culture solution can be utilized as a rooting promoter or a plant growth regulator.
  • Example 14 Bacillus thuringiensis B-18 strain (reception number NITE ABP-858) was cultured in 10 mL of NB at 27 ° C. for 3 days, and the culture was centrifuged at 15,000 rpm for 10 minutes. After discarding the supernatant, 10 mL of sterilized water was added, suspended well, and centrifuged again to wash the cells. This washing was repeated two more times, and 10 mL of sterilized water was added and suspended sufficiently to obtain an inoculation source.
  • tomato variety House Momotaro (Takii seedling) seeds are immersed in 80% ethanol for 1 minute, then washed with sterile water, then immersed in 1% sodium hypochlorite for 5 minutes, and then twice with sterile water. After performing seed disinfection by washing, the seed was sown on the surface of a medium containing 1% by mass of pre-sterilized gellite (manufactured by Wako Pure Chemical Industries, Ltd.). After culturing at 27 ° C.

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Abstract

Disclosed is a novel method for producing an indolylalkyl succinic acid amide compound, which is useful as a plant growth accelerator, by microorganisms. Specifically disclosed is a method for producing an indolylalkyl succinic acid amide compound represented by general formula (2) (wherein R1, R2 and n are as defined below) or a salt thereof, which is characterized in that microorganisms belonging to the genus Bacillus and having the power of synthesizing an indolylalkyl succinic acid amide are cultured in a medium containing an indolylalkyl amine represented by general formula (1) (wherein R1 and R2 each independently represents a hydrogen atom, a halogen atom, a hydroxy group, an alkoxy group or an alkyl group; R3 represents a hydrogen atom or a carboxyl group; and n represents a number of 0-3) or a salt thereof.

Description

インドリルアルキルコハク酸アミド化合物の製造法Method for producing indolylalkyl succinamide compounds
　本発明は、植物成長調整剤として有用なインドリルアルキルコハク酸アミド化合物の微生物を利用した製造法に関する。 The present invention relates to a process for producing an indolylalkyl succinamide compound useful as a plant growth regulator using a microorganism.
　発根促進活性が高く、植物の成長を促進する成分は、農業及び林業分野において強く望まれており、数多くの研究がなされている。そのような状況下、下記一般式（ａ） Ingredients that have high rooting promoting activity and promote plant growth are strongly desired in the fields of agriculture and forestry, and many studies have been made. Under such circumstances, the following general formula (a)
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
（式中、Ａｒは置換基を有していてもよいフェニル、ナフチル又はインドリル基を示し、ｎは０～３の数を示す）
で表されるコハク酸アミド化合物又はその塩が優れた発根促進作用を有し、植物成長調整剤として有用であることが知られている（特許文献１及び２）。 (In the formula, Ar represents an optionally substituted phenyl, naphthyl or indolyl group, and n represents a number of 0 to 3)
It is known that a succinamide compound represented by the formula (1) or a salt thereof has an excellent rooting promoting action and is useful as a plant growth regulator (Patent Documents 1 and 2).
　このコハク酸アミド化合物は特許文献１及び２記載のように化学合成によって得ることもできるが、このうちフェネチルコハク酸アミドはバチルス属に属する微生物によっても生産されることが知られている（非特許文献１）。 This succinic acid amide compound can be obtained by chemical synthesis as described in Patent Documents 1 and 2, and phenethyl succinic acid amide is known to be produced by microorganisms belonging to the genus Bacillus (non-patent). Reference 1).
特開平１－２５５６０７号公報JP-A-1-255607 特開２００１－１３９４０５号公報JP 2001-139405 A
　現在、市場に流通している植物成長調整剤のほとんどは化学合成によって製造されているが、消費者の間には物質自体の安全性に疑念をもつ風潮も未だに強い。このことは、有機農産物や減農薬栽培農産物がもてはやされていることからも裏付けられる。この点を鑑みると、植物成長調整剤を含む農薬としては、天然由来のものが好ましく、化学合成により得られるものでなく、微生物によって生産されるものが望まれている。
　しかしながら、インドリルアルキルコハク酸アミドについての微生物による生産法についての報告はない。
　従って、本発明の課題は、植物成長促進剤として有用なインドリルアルキルコハク酸アミド化合物の微生物による新たな製造法を提供することにある。 Currently, most plant growth regulators on the market are produced by chemical synthesis, but there is still a strong tendency among consumers to question the safety of the substance itself. This is supported by the fact that organic agricultural products and agricultural products with reduced agricultural chemicals are being used. In view of this point, a pesticide containing a plant growth regulator is preferably derived from nature, and is not obtained by chemical synthesis but is produced by a microorganism.
However, there is no report about the production method by microorganisms about indolyl alkyl succinamide.
Accordingly, an object of the present invention is to provide a novel method for producing an indolylalkylsuccinamide compound useful as a plant growth promoter by a microorganism.
　そこで本発明者は、インドリルアルキルコハク酸アミドの生産能を有する新たな微生物を探索したところ、バチルス属に属する微生物がトリプタミン、トリプトファン及びセロトニン塩酸塩からインドリルアルキルコハク酸アミド化合物を効率良く生産することを見出し、本発明を完成した。 Therefore, the present inventor searched for a new microorganism capable of producing indolylalkylsuccinic acid amide, and microorganisms belonging to the genus Bacillus efficiently produced indolylalkylsuccinic acid amide compounds from tryptamine, tryptophan and serotonin hydrochloride. The present invention has been completed.
　すなわち、本発明は、一般式（１） That is, the present invention has the general formula (1)
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
（式中、Ｒ1及びＲ2はそれぞれ独立して水素原子、ハロゲン原子、水酸基、アルコキシ基又はアルキル基を示し、Ｒ3は水素原子又はカルボキシル基を示し、ｎは０～３の数を示す）
で表されるインドリルアルキルアミン類又はその塩を含有する培地中で、インドリルアルキルコハク酸アミド合成能を有するバチルス属に属する微生物を培養することを特徴とする、一般式（２） (Wherein R 1 and R 2 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group or an alkyl group, R 3 represents a hydrogen atom or a carboxyl group, and n represents a number of 0 to 3) )
A microorganism belonging to the genus Bacillus having the ability to synthesize indolylalkylsuccinic acid amide is cultured in a medium containing the indolylalkylamines or salts thereof represented by the general formula (2)
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
（式中、Ｒ1、Ｒ2及びｎは前記と同じ）
で表されるインドリルアルキルコハク酸アミド化合物又はその塩の製造法を提供するものである。 (Wherein R 1 , R 2 and n are the same as above)
The manufacturing method of the indolyl alkyl succinic acid amide compound represented by these, or its salt is provided.
　本発明方法によれば、バチルス属に属する微生物の培養により、植物成長調整剤として有用なインドリルアルキルコハク酸アミド化合物又はその塩が効率良く得られる。 According to the method of the present invention, an indolylalkylsuccinamide compound or a salt thereof useful as a plant growth regulator can be efficiently obtained by culturing a microorganism belonging to the genus Bacillus.
　本明細書において、質量％とは(wt／vol)％を意味し、容量％とは(vol／vol)％を意味する。 In this specification, mass% means (wt / vol)%, and volume% means (vol / vol)%.
　本発明のインドリルアルキルコハク酸アミド化合物の製造法に用いられる微生物は、バチルス・チューリンゲンシス（Bacillus thuringiensis）、バチルス・セレウス（Bacillus cereus）、バチルス・ズブチリス（Bacillus subtilis）、バチルス・アシディセラー（Bacillus acidiceler）、バチルス・アシディコーラ（Bacillus acidicola）、バチルス・アエオリウス（Bacillus aeolius）、バチルス・アエリウス（Bacillus aerius）、バチルス・アエロフィルス（Bacillus aerophilus）、バチルス・アガラドヘレンス（Bacillus agaradhaerens）、バチルス・アキバイ（Bacillus akibai）、バチルス・アルカリフィルス（Bacillus alcalophilus）、バチルス・アルギコーラ（Bacillus algicola）、バチルス・アルカリジアゾトロフィカス（Bacillus alkalidiazotrophicus）、バチルス・アルカリテルリス（Bacillus alkalitelluris）、バチルス・アルティチュジニス（Bacillus altitudinis）、バチルス・アルベアユエンシス（Bacillus alveayuensis）、バチルス・アミロリクエファシエンス（Bacillus amyloliquefaciens）、バチルス・アンスラシス（Bacillus anthracis）、バチルス・アクイマリス（Bacillus aquimaris）、バチルス・アレノシイ（Bacillus arenosi）、バチルス・アルセニシセレナティス（Bacillus arseniciselenatis）、バチルス・アルセニカス（Bacillus arsenicus）、バチルス・アーヴァイ（Bacillus arvi）、バチルス・アサヒイ（Bacillus asahii）、バチルス・アトロファエウス（Bacillus atrophaeus）、バチルス・アウランティアカス（Bacillus aurantiacus）、バチルス・アキサクィエンシス（Bacillus axarquiensis）、バチルス・アゾトフォーマンス（Bacillus azotoformans）、 Microorganisms used in the method for producing an indolylalkylsuccinamide compound of the present invention include Bacillus thuringiensis , Bacillus cereus , Bacillus subtilis , Bacillus acidiceler. ), Bacillus Ashidikora (Bacillus acidicola), Bacillus Aeoriusu (Bacillus aeolius), Bacillus Aeriusu (Bacillus aerius), Bacillus Aerofirusu (Bacillus aerophilus), Bacillus Agaradoherensu (Bacillus agaradhaerens), Bacillus Akibai (Bacillus akibai) , Bacillus alkaline Filth (Bacillus alcalophilus), Bacillus Arugikora (Bacillus algicola), Bacillus alkaline diazo Toro Ficus (Bacillus alkalidiazotrophicus), Bacillus Arukariteruri Scan (Bacillus alkalitelluris), Bacillus Ultimatum Ju Hee scan (Bacillus altitudinis), Bacillus Al Bear Yuen cis (Bacillus alveayuensis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus anthracis (Bacillus anthracis), Bacillus Akuimarisu ( Bacillus aquimaris), Bacillus Arenoshii (Bacillus arenosi), Bacillus Arce Western Serena Sevilla (Bacillus arseniciselenatis), Bacillus Arusenikasu (Bacillus arsenicus), Bacillus Avai (Bacillus arvi), Bacillus Asahii (Bacillus asahii), Bacillus atrophaeus (Bacillus atrophaeus), Bacillus A uranium tier dregs (Bacillus aurantiacus), Bacillus Aki Saku I ene cis (Bacillus axarquiensis), Bacillus azo door performance (Bacillus azotoformans),
バチルス・バディウス（Bacillus badius）、バチルス・バーバリカス（Bacillus barbaricus）、バチルス・バタビエンシス（Bacillus bataviensis）、バチルス・ベンゾボランス（Bacillus benzoevorans）、バチルス・ボゴリエンシス（Bacillus bogoriensis）、バチルス・ボロニフィルス（Bacillus boroniphilus）、バチルス・ブタノリボランス（Bacillus butanolivorans）、バチルス・カーボニフィルス（Bacillus carboniphilus）、バチルス・セセンベンシス（Bacillus cecembensis）、バチルス・セルロシリティカス（Bacillus cellulosilyticus）、バチルス・チャガノレンシス（Bacillus chagannorensis）、バチルス・シービー（Bacillus cibi）、バチルス・サーキュランス（Bacillus circulans）、バチルス・クラーキー（Bacillus clarkii）、バチルス・クラウシー（Bacillus clausii）、バチルス・コアギュランス（Bacillus coagulans）、バチルス・コアウイレンシス（Bacillus coahuilensis）、バチルス・コーニー（Bacillus cohnii）、バチルス・デシシフロンディス（Bacillus decisifrondis）、バチルス・デコロラチオニス（Bacillus decolorationis）、バチルス・ドレンテンシス（Bacillus drentensis）、バチルス・エダフィカス（Bacillus edaphicus）、バチルス・エンドフィティカス（Bacillus endophyticus）、バチルス・ファラギニス（Bacillus farraginis）、バチルス・フォスティディオサス（Bacillus fastidiosus）、バチルス・ファルマス（Bacillus firmus）、バチルス・フレクサス（Bacillus flexus）、バチルス・ホラミニス（Bacillus foraminis）、バチルス・フォルディ（Bacillus fordii）、バチルス・フォルティス（Bacillus fortis）、バチルス・フマリオリィ（Bacillus fumarioli）、バチルス・フニクラス（Bacillus funiculus）、バチルス・ガラクトシディリティカス（Bacillus galactosidilyticus）、バチルス・ゲラティニ（Bacillus gelatini）、バチルス・ギブソニー（Bacillus gibsonii）、バチルス・ハルマパラス（Bacillus halmapalus）、バチルス・ハロデュランス（Bacillus halodurans）、バチルス・ハロフィルス（Bacillus halophilus）、バチルス・ヘミセルロシリティカス（Bacillus hemicellulosilyticus）、バチルス・ハーバーステイネンシス（Bacillus herbersteinensis）、 Bacillus badius (Bacillus badius), Bacillus Babarikasu (Bacillus barbaricus), Bacillus Batabienshisu (Bacillus bataviensis), Bacillus Benzoboransu (Bacillus benzoevorans), Bacillus Bogorienshisu (Bacillus bogoriensis), Bacillus Boronifirusu (Bacillus boroniphilus), Bacillus Butanoriboransu (Bacillus butanolivorans), Bacillus Kabonifirusu (Bacillus carboniphilus), Bacillus Sesenbenshisu (Bacillus cecembensis), Bacillus cell Russia utility dregs (Bacillus cellulosilyticus), Bacillus Chaganorenshisu (Bacillus chagannorensis), Bacillus CB (Bacillus cibi) , Bacillus circulans , Bacillus clarkii , Bacillus clausii, Bacillus colus Agyuransu (Bacillus coagulans), Bacillus Koauirenshisu (Bacillus coahuilensis), Bacillus Coney (Bacillus cohnii), Bacillus de Shishi CFC disk (Bacillus decisifrondis), Bacillus Dekororachionisu (Bacillus decolorationis), Bacillus Dorentenshisu (Bacillus drentensis), Bacillus · Edafikasu (Bacillus edaphicus), Bacillus end Fi Atlantica scan (Bacillus endophyticus), Bacillus Faraginisu (Bacillus farraginis), Bacillus follower stay Dio suspension (Bacillus fastidiosus), Bacillus Falmouth (Bacillus firmus), Bacillus Furekusasu (Bacillus flexus ), Bacillus foraminis , Bacillus fordii , Bacillus fortis , Bacillus fumariol i), Bacillus Funikurasu (Bacillus funiculus), Bacillus galactosyl di utility dregs (Bacillus galactosidilyticus), Bacillus Geratini (Bacillus gelatini), Bacillus Gibusoni (Bacillus gibsonii), Bacillus Harumaparasu (Bacillus halmapalus), Bacillus halodurans (Bacillus halodurans), Bacillus Harofirusu (Bacillus halophilus), Bacillus hemi-cell Russia utility dregs (Bacillus hemicellulosilyticus), Bacillus Harbor Stay Nene cis (Bacillus herbersteinensis),
バチルス・ホリコシー（Bacillus horikoshii）、バチルス・ホルティ（Bacillus horti）、バチルス・フミ（Bacillus humi）、バチルス・ファジンポエンシス（Bacillus hwajinpoensis）、バチルス・イドリエンシス（Bacillus idriensis）、バチルス・インディカス（Bacillus indicus）、バチルス・インファンティス（Bacillus infantis）、バチルス・インファーナス（Bacillus infernus）、バチルス・インソリタス（Bacillus insolitus）、バチルス・イサベリエ（Bacillus isabeliae）、バチルス・チョッカリ（Bacillus jeotgali）、バチルス・コリエンシス（Bacillus koreensis）、バチルス・クルーウィッチアエ（Bacillus krulwichiae）、バチルス・レヘンシス（Bacillus lehensis）、バチルス・レンタス（Bacillus lentus）、バチルス・リケニホルミス（Bacillus licheniformis）、バチルス・リトラリス（Bacillus litoralis）、バチルス・ルシフェレンシス（Bacillus luciferensis）、バチルス・マカウエンシス（Bacillus macauensis）、バチルス・マキアエ（Bacillus macyae）、バチルス・マラシテンシス（Bacillus malacitensis）、バチルス・マンナニリティカス（Bacillus mannanilyticus）、バチルス・マリナス（Bacillus marinus）、バチルス・マリスフラビィ（Bacillus marisflavi）、バチルス・マシリエンシス（Bacillus massiliensis）、バチルス・メガテリウム（Bacillus megaterium）、バチルス・メタノリカス（Bacillus methanolicus）、バチルス・モジャベンシス（Bacillus mojavensis）、バチルス・ムシラギノサス（Bacillus mucilaginosus）、バチルス・ムラリス（Bacillus muralis）、バチルス・ムリマルティニ（Bacillus murimartini）、バチルス・ミコイデス（Bacillus mycoides）、バチルス・ネールソニー（Bacillus nealsonii）、バチルス・ニアベンシス（Bacillus niabensis）、バチルス・ニアシニ（Bacillus niacini）、バチルス・ノバリス（Bacillus novalis）、バチルス・オデッセイ（Bacillus odysseyi）、バチルス・オクヘンシス（Bacillus okhensis）、バチルス・オクヒデンシス（Bacillus okuhidensis）、バチルス・オレロニウス（Bacillus oleronius）、バチルス・オシメンシス（Bacillus oshimensis）、バチルス・パリダス（Bacillus pallidus）、バチルス・パナシテラエ（Bacillus panaciterrae）、バチルス・パタゴニエンシス（Bacillus patagoniensis）、バチルス・プラコルチディス（Bacillus plakortidis）、バチルス・ポチェオネンシス（Bacillus pocheonensis）、バチルス・ポリゴニ（Bacillus polygoni）、 Bacillus Horikoshi (Bacillus horikoshii), Bacillus Horuti (Bacillus horti), Bacillus Fumi (Bacillus humi), Bacillus file Jin Po-ene-cis (Bacillus hwajinpoensis), Bacillus Idorienshisu (Bacillus idriensis), Bacillus indicus (Bacillus indicus) , Bacillus infantis (Bacillus infantis), Bacillus in the furnace (Bacillus infernus), Bacillus Insoritasu (Bacillus insolitus), Bacillus Isaberie (Bacillus isabeliae), Bacillus Chokkari (Bacillus jeotgali), Bacillus Korienshisu (Bacillus koreensis ), Bacillus krulwichiae , Bacillus lehensis , Bacillus lentus , Bacillus licheniformis , Bacillus licheniformis Torarisu (Bacillus litoralis), Bacillus Rushiferenshisu (Bacillus luciferensis), Bacillus Makauenshisu (Bacillus macauensis), Bacillus Makiae (Bacillus macyae), Bacillus Marashitenshisu (Bacillus malacitensis), Bacillus Man Nani utility dregs (Bacillus mannanilyticus) , Bacillus marinus (Bacillus marinus), Bacillus Marisufurabyi (Bacillus marisflavi), Bacillus Mashirienshisu (Bacillus massiliensis), Bacillus megaterium (Bacillus megaterium), Bacillus methanolicus (Bacillus methanolicus), Bacillus Mojabenshisu (Bacillus mojavensis), Bacillus · Mushiraginosasu (Bacillus mucilaginosus), Bacillus Murarisu (Bacillus muralis), Bacillus Muri Martini (Bacillus murimartini), Bacillus mycoides (Bacillus mycoides , Bacillus Nehru Sony (Bacillus nealsonii), Bacillus two Avensis (Bacillus niabensis), Bacillus Niashini (Bacillus niacini), Bacillus Novalis (Bacillus novalis), Bacillus Odyssey (Bacillus odysseyi), Bacillus Okuhenshisu (Bacillus okhensis) , Bacillus Okuhidenshisu (Bacillus okuhidensis), Bacillus Oreroniusu (Bacillus oleronius), Bacillus Oshimenshisu (Bacillus oshimensis), Bacillus Paridasu (Bacillus pallidus), Bacillus Panashiterae (Bacillus panaciterrae), Bacillus Patagonienshisu (Bacillus patagoniensis), Bacillus・Placortidis ( Bacillus plakortidis ), Bacillus pocheonensis ( Bacillus pocheonensis ), Bacillus polygoni ( Bacillus polygoni ),
バチルス・シュードアルカリフィルス（Bacillus pseudalcaliphilus）、バチルス・シュードファーマス（Bacillus pseudofirmus）、バチルス・シュードミコイデス（Bacillus pseudomycoides）、バチルス・シクロデュランス（Bacillus psychrodurans）、バチルス・フィクロサッカロリティカス（Bacillus psychrosaccharolyticus）、バチルス・フィクロトレランス（Bacillus psychrotolerans）、バチルス・プミラス（Bacillus pumilus）、バチルス・ピクナス（Bacillus pycnus）、バチルス・キングダオネンシス（Bacillus qingdaonensis）、バチルス・ルリス（Bacillus ruris）、バチルス・サフェンシス（Bacillus safensis）、バチルス・サラリウス（Bacillus salarius）、バチルス・サリフィルス（Bacillus saliphilus）、バチルス・シュリジェリー（Bacillus schlegelii）、バチルス・セレナターセナティス（Bacillus selenatarsenatis）、バチルス・セレニティレドセンス（Bacillus selenitireducens）、バチルス・セオハエアネンシス（Bacillus seohaeanensis）、バチルス・サックレトニー（Bacillus shackletonii）、バチルス・シルベストリス（Bacillus silvestris）、バチルス・シンプレックス（Bacillus simplex）、バチルス・シラリス（Bacillus siralis）、バチルス・スミシー（Bacillus smithii）、バチルス・ソリ（Bacillus soli）、バチルス・ソノレンシス（Bacillus sonorensis）、バチルス・スフェリカス（Bacillus sphaericus）、バチルス・スポロサーモデュランス（Bacillus sporothermodurans）、バチルス・ストラトスフェリカス（Bacillus stratosphericus）、バチルス・サブテラネウス（Bacillus subterraneus）、バチルス・タエアネンシス（Bacillus taeanensis）、バチルス・テクィレンシス（Bacillus tequilensis）、バチルス・サーマンタルクティカス（Bacillus thermantarcticus）、バチルス・サーモアミロボランス（Bacillus thermoamylovorans）、バチルス・サーモクローカー（Bacillus thermocloacae）、バチルス・チオパランス（Bacillus thioparans）、バチルス・ツスシー（Bacillus tusciae）、バチルス・バリスモーチス（Bacillus vallismortis）、バチルス・ベデリ（Bacillus vedderi）、バチルス・ベレゼンシス（Bacillus velezensis）、バチルス・ビエトナメンシス（Bacillus vietnamensis）、バチルス・ビレティ（Bacillus vireti）、バチルス・ワコエンシス（Bacillus wakoensis）、バチルス・ヴェイヘンステファネンシス（Bacillus weihenstephanensis）に属し、インドリルアルキルコハク酸アミド合成能を有するものであれば特に制限されないが、インドリルアルキルコハク酸アミド化合物合成能の点で、バチルス・チューリンゲンシス、バチルス・セレウス、バチルス・ズブチリス、バチルス・メガテリウムに属するものが特に好ましい。 Bacillus pseudalcaliphilus , Bacillus pseudofirmus , Bacillus pseudomycoides , Bacillus psychrodurans , Bacillus physaccharolyticus , Bacillus Fi black tolerance (Bacillus psychrotolerans), Bacillus pumilus (Bacillus pumilus), Bacillus Pikunasu (Bacillus pycnus), Bacillus King Dao Nene cis (Bacillus qingdaonensis), Bacillus Rurisu (Bacillus ruris), Bacillus Safenshisu ( Bacillus safensis), Bacillus Sarariusu (Bacillus salarius), Bacillus Sarifirusu (Bacillus saliphilus), Bacillus Sri Jerry (Bacillus schlegelii), Bacillus Serenatasena Office (Bacillus selenatarsenatis), Bacillus Serenity TOLEDO sense (Bacillus selenitireducens), Bacillus Theo Ha air Nene cis (Bacillus seohaeanensis), Bacillus Sakkuretoni (Bacillus shackletonii), Bacillus sylvestris (Bacillus silvestris), Bacillus simplex ( Bacillus simplex ), Bacillus siralis , Bacillus smithii , Bacillus soli , Bacillus sonorensis , Bacillus sphaericus , Bacillus sphaericus (Bacillus sporothermodurans), Bacillus Stratos FeliCa scan (Bacillus stratosphericus), Bacillus Sabuteraneusu (Bacillus subterraneus), Bacillus Taeanenshisu (Bacillus taeanensis), Bacillus Tekui Nshisu (Bacillus tequilensis), Bacillus Thurman talc Atlantica scan (Bacillus thermantarcticus), Bacillus thermo-Ami Robo lance (Bacillus thermoamylovorans), Bacillus thermo-crore car (Bacillus thermocloacae), Bacillus Chioparansu (Bacillus thioparans), Bacillus Tsusushi (Bacillus tusciae), Bacillus Barisumochisu (Bacillus vallismortis), Bacillus Bederi (Bacillus vedderi), Bacillus Berezenshisu (Bacillus velezensis), Bacillus Bietonamenshisu (Bacillus vietnamensis), Bacillus Bireti (Bacillus vireti), Bacillus Wakoenshisu (Bacillus wakoensis) Is not particularly limited as long as it belongs to Bacillus weihenstephanensis and has the ability to synthesize indolylalkylsuccinic acid amides. Particularly preferred are those belonging to Bacillus thuringiensis, Bacillus cereus, Bacillus subtilis, Bacillus megaterium in terms of the ability to synthesize rualkylsuccinamide compounds.
　上記のうち好適な具体例としては、バチルス・チューリンゲンシスＪＣＭ２０３８６株、同Ｂ－２株、同Ｂ－３（郵便番号292-0818 日本国 千葉県 木更津市 かずさ鎌足2-5-8（独）製品評価技術基板機構特許微生物寄託センター 受領番号ＮＩＴＥ ＡＰ－６８９）株、同Ｂ－９株、同Ｂ－１０株、同Ｂ－１８（ＮＩＴＥ ＡＢＰ－８５８）株、同Ｂ－２１株、同Ｂ－２２株、同Ｂ－２４株（ＮＩＴＥ ＡＰ－８５７）、同Ｂ－２６株、同Ｂ－３３株、同Ｂ－３６株、同Ｂ－５９株；バチルス・セレウスＢ－５株、同Ｂ－６（ＮＩＴＥ ＡＰ－６９０）株、同Ｂ－７株、同Ｂ－８株、同Ｂ－１１株、同Ｂ－１２株、同Ｂ－１４株、同Ｂ－１５株、同Ｂ－１６株（ＮＩＴＥ ＡＰ－８５６）；バチルス・メガテリウムＢ－３２（ＮＩＴＥ ＡＢＰ－８５９）株；バチルス・ズブチリスＪＣＭ１４６５株等が挙げられ、インドリルアルキルコハク酸アミド化合物合成能の点で、バチルス・チューリンゲンシスＪＣＭ２０３８６株、同Ｂ－３株、同Ｂ－１８株、同Ｂ－２４株；バチルス・セレウスＢ－６株、同Ｂ－１５株、同Ｂ－１６株；バチルス・ズブチリスＪＣＭ１４６５株が特に好ましい。
  なお、寄託日は受領番号ＮＩＴＥ　ＡＰ－６８９株・ＮＩＴＥ ＡＰ－６９０株は平成２０年（２００８）年１２月５日であり、受領番号ＮＩＴＥ　ＡＰ－８５６・ＡＰ－８５７・ＡＢＰ－８５８・ＡＢＰ－８５９は平成２１（２００９）年１２月２５日である。
　上記のうち、バチルス・チューリンゲンシスＪＣＭ２０３８６株及びバチルス・ズブチリスＪＣＭ１４６５株は、独立行政法人　理化学研究所から購入できる菌株である。またその他の菌株は、土壌から分離されたものである。 Preferred examples of the above include Bacillus thuringiensis JCM20386, B-2 and B-3 (postal code 292-0818 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-689), B-9, B-10, B-18 (NITE ABP-858), B-21, B -22, B-24 (NITE AP-857), B-26, B-33, B-36, B-59; Bacillus cereus B-5, B -6 (NITE AP-690), B-7, B-8, B-11, B-12, B-14, B-15, B-16 Strain (NITE AP-856); Bacillus megaterium B-32 (NITE ABP-859) strain Bacillus subtilis JCM1465 strain and the like. In terms of the ability to synthesize indolylalkylsuccinic acid amide compounds, Bacillus thuringiensis JCM20386 strain, B-3 strain, B-18 strain, B-24 strain; Cereus B-6 strain, B-15 strain, B-16 strain; Bacillus subtilis JCM1465 strain is particularly preferable.
The deposit date is NITE AP-689 shares and NITE AP-690 shares are December 5, 2008, and the receipt numbers NITE AP-856, AP-857, ABP-858, ABP- 859 is December 25, 2009.
Among the above, Bacillus thuringiensis JCM20386 strain and Bacillus subtilis JCM1465 strain are strains that can be purchased from RIKEN. Other strains were isolated from soil.
　これらの微生物を用いて一般式（２）のインドリルアルキルコハク酸アミド化合物又はその塩を製造するには、培地中に一般式（１）で表されるインドリルアルキルアミン類又はその塩を含有せしめる。 In order to produce an indolylalkylsuccinic acid amide compound of the general formula (2) or a salt thereof using these microorganisms, the medium contains an indolylalkylamine or a salt thereof represented by the general formula (1) Let me.
　Ｒ1及びＲ2としては、水素原子、ハロゲン原子、水酸基、Ｃ1-6アルキル基、Ｃ1-6アルコキシ基が挙げられる。ここでＣ1-6アルキル基としては、メチル基、エチル基、イソプロピル基、ｔ－ブチル基等が挙げられる。Ｃ1-6アルコキシ基としては、メトキシ基、エトキシ基、イソプロピルオキシ基等が挙げられる。またハロゲン原子としては、フッ素原子、塩素原子、臭素原子、ヨウ素原子が挙げられる。 Examples of R 1 and R 2 include a hydrogen atom, a halogen atom, a hydroxyl group, a C 1-6 alkyl group, and a C 1-6 alkoxy group. Here, examples of the C 1-6 alkyl group include a methyl group, an ethyl group, an isopropyl group, and a t-butyl group. Examples of the C 1-6 alkoxy group include a methoxy group, an ethoxy group, and an isopropyloxy group. Moreover, as a halogen atom, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom are mentioned.
　一般式（１）及び（２）中、ｎは０～３の数を示すが、０～２、特に１が好ましい。 In general formulas (1) and (2), n represents a number from 0 to 3, with 0 to 2, particularly 1 being preferred.
　一般式（１）及び（２）中、Ｒ1及びＲ2としては水素原子、水酸基が特に好ましく、ｎは１が特に好ましい。 In general formulas (1) and (2), R 1 and R 2 are particularly preferably a hydrogen atom or a hydroxyl group, and n is particularly preferably 1.
　インドリルアルキルアミン類（１）としては、インドリルアルキルアミン類（Ｒ3＝Ｈ）でもよいし、インドリルアルキルカルボキシアミン類（Ｒ3＝ＣＯＯＨ）でもよいが、トリプタミン（Ｒ1＝Ｈ、Ｒ2＝Ｈ、ｎ＝１、Ｒ3＝Ｈ）、トリプトファン（Ｒ1＝Ｈ、Ｒ2＝Ｈ、ｎ＝１、Ｒ3＝ＣＯＯＨ）、セロトニン（Ｒ1＝ＯＨ、Ｒ2＝Ｈ、ｎ＝１、Ｒ3＝ＣＯＯＨ，若しくはＲ1＝Ｈ、Ｒ2＝ＯＨ、ｎ＝１、Ｒ3＝Ｈ）、あるいはこれらの塩が収率向上の点で特に好ましく、これらの化合物は２種以上を併用してもよい。
　なお、インドリルアルキルアミン類（１）の塩としては、塩酸塩、硝酸塩、硫酸塩等が挙げられるが、塩酸塩が好ましい。 Indolylalkylamines (1) may be indolylalkylamines (R 3 = H) or indolylalkylcarboxyamines (R 3 = COOH), but tryptamine (R 1 = H, R 2 = H, n = 1, R 3 = H), tryptophan (R 1 = H, R 2 = H, n = 1, R 3 = COOH), serotonin (R 1 = OH, R 2 = H, n = 1, R 3 = COOH, or R 1 = H, R 2 = OH, n = 1, R 3 = H), or a salt thereof is particularly preferable in terms of yield improvement. You may use together.
In addition, examples of the salt of indolylalkylamines (1) include hydrochloride, nitrate, sulfate and the like, and hydrochloride is preferable.
　培地中のインドリルアルキルアミン類（１）又はその塩の濃度は、インドリルアルキルコハク酸アミド化合物の生産性の点から０．００１～１０質量％、さらに０．００１～５質量％、特に０．０１～５質量％が好ましい。また、当該インドリルアルキルアミン類（１）は、反応系に一括又は連続的に加えることができる。 The concentration of the indolylalkylamines (1) or a salt thereof in the medium is 0.001 to 10% by mass, more preferably 0.001 to 5% by mass, particularly 0 in terms of productivity of the indolylalkylsuccinic acid amide compound. 0.01 to 5% by mass is preferable. The indolylalkylamines (1) can be added to the reaction system all at once or continuously.
　培地中にはマンガンイオン（Ｍｎ2+）を含有させることによりインドリルアルキルコハク酸アミド化合物をさらに効率よく生産させることができる。マンガンイオンを添加する場合は硫酸マンガン、塩酸マンガン、炭酸マンガン、ＥＤＴＡマンガン等いずれでも可能であり、これらを１種又は２種以上用いることができるが、硫酸マンガンが特に好ましい。
　培地中の含有量はマンガンイオン（Ｍｎ2+）として１～２０００ｐｐｍ、特に２～１０００ｐｐｍが好ましい。なお、マンガンイオン（Ｍｎ2+）は、反応系に一括又は連続的に加えることができる。 By containing manganese ions (Mn 2+ ) in the medium, an indolylalkyl succinamide compound can be produced more efficiently. When manganese ions are added, any of manganese sulfate, manganese hydrochloride, manganese carbonate, EDTA manganese, and the like can be used, and one or more of these can be used, and manganese sulfate is particularly preferable.
The content in the medium is preferably 1 to 2000 ppm, particularly 2 to 1000 ppm as manganese ions (Mn 2+ ). Manganese ions (Mn 2+ ) can be added to the reaction system all at once or continuously.
　また、培地中には有機酸又はその塩を含有させることによりインドリルアルキルコハク酸アミド化合物をさらに効率よく生産させることができる。有機酸としてはリンゴ酸、フマル酸、コハク酸、クエン酸、イソクエン酸、α－ケトグルタル酸、オキサロ酢酸等が挙げられ、これらを１種又は２種以上用いることができる。このうち、インドリルアルキルコハク酸アミド化合物産生効率の点で、リンゴ酸、フマル酸、コハク酸が好ましく、Ｌ－リンゴ酸、Ｄ－リンゴ酸、フマル酸がより好ましい。
　これらの有機酸の培地中の濃度は０．１～１０質量％が好ましく、０．２～１質量％がより好ましい。なお、有機酸又はその塩は、反応系に一括又は連続的に加えることができる。 Moreover, an indolyl alkyl succinamide compound can be produced more efficiently by containing an organic acid or a salt thereof in the medium. Examples of the organic acid include malic acid, fumaric acid, succinic acid, citric acid, isocitric acid, α-ketoglutaric acid, oxaloacetic acid, and the like, and one or more of these can be used. Of these, malic acid, fumaric acid, and succinic acid are preferable, and L-malic acid, D-malic acid, and fumaric acid are more preferable in terms of production efficiency of indolylalkyl succinic acid amide compounds.
The concentration of these organic acids in the medium is preferably 0.1 to 10% by mass, more preferably 0.2 to 1% by mass. The organic acid or a salt thereof can be added to the reaction system all at once or continuously.
　本発明の製造法において、前記微生物の培養は、インドリルアルキルアミン類（１）又はその塩、必要に応じてその他上記の化合物を培地に添加する以外は、通常のバチルス属微生物が増殖する条件で行えばよい。
　培地には、バチルス属微生物の増殖に必要な炭素源、窒素源、ビタミン源、ミネラル源等を添加するのが好ましい。炭素源としては、グルコース、水飴、可溶性デンプンなどが用いられる。窒素源としては、トリプトン、ソイトン、酵母エキス、ペプトン、カゼイン、カゼイン消化物、肉エキス、コーンスチープリカー、アミノ酸液、大豆粕、硫酸アンモニウム、塩化アンモニウム等を用いることができるが、トリプトファン含量の高いカゼイン、カゼイン消化物、酵母エキス、コーンスチープリカー等が好ましいことは言うまでもない。
　ミネラル源としては、マグネシウム塩、カリウム塩、リン酸塩、鉄塩などを用いることができる。また、これらを総合的に含有する培地としては、牛乳；チーズホエイ、大豆ホエイ等のホエイ等を用いることができる。培養方法としては、液体培地を用いて、振盪培養、撹拌培養を行えばよい。 In the production method of the present invention, the microorganism is cultured under the conditions under which normal microorganisms of the genus Bacillus grow, except that indolylalkylamines (1) or a salt thereof and, if necessary, other compounds described above are added to the medium. Just do it.
It is preferable to add a carbon source, a nitrogen source, a vitamin source, a mineral source and the like necessary for the growth of Bacillus microorganisms to the medium. As the carbon source, glucose, starch syrup, soluble starch and the like are used. As the nitrogen source, tryptone, soyton, yeast extract, peptone, casein, casein digest, meat extract, corn steep liquor, amino acid solution, soybean cake, ammonium sulfate, ammonium chloride, etc. can be used, but casein with a high tryptophan content. Needless to say, casein digest, yeast extract, corn steep liquor and the like are preferable.
As the mineral source, magnesium salt, potassium salt, phosphate, iron salt and the like can be used. Moreover, as a culture medium containing these comprehensively, whey, such as milk; cheese whey, soybean whey, etc. can be used. As a culture method, a shaking culture and a stirring culture may be performed using a liquid medium.
　培養条件としては好気的条件で、８～４５℃、ｐＨ３．２～９．５が好ましい。培養時間は、２４～７２０時間でよい。 The culture conditions are aerobic conditions, preferably 8 to 45 ° C. and pH 3.2 to 9.5. The culture time may be 24 to 720 hours.
　前記培養により、培養液中にインドリルアルキルコハク酸アミド化合物（２）又はその塩が蓄積するので、常法によりインドリルアルキルコハク酸アミド化合物（２）又はその塩を採取すればよい。この培養液は、そのまま植物成長調整剤や肥料として用いることもできる。また、イオン交換樹脂、多孔性合成吸着剤、溶媒抽出等によってインドリルアルキルコハク酸アミド化合物（２）又はその塩を部分精製又は精製単離して利用することもできる。 Since the indolylalkyl succinamide compound (2) or a salt thereof accumulates in the culture medium by the culture, the indolylalkyl succinamide compound (2) or a salt thereof may be collected by a conventional method. This culture solution can also be used as a plant growth regulator or fertilizer as it is. The indolylalkylsuccinic acid amide compound (2) or a salt thereof can be partially purified or purified and isolated by ion exchange resin, porous synthetic adsorbent, solvent extraction, or the like.
　例えば、イオン交換樹脂による部分精製物は、上記培養液のｐＨを中性領域（ｐＨ５～８）に調整後、強塩基性イオン交換樹脂又は弱塩基性イオン交換樹脂に吸着させた後、酸含有アルコール水溶液で溶出して得ることができる。このとき、吸着前のイオン交換樹脂を蟻酸型又は酢酸型等に置換することが好ましい。
　当該溶液の酸濃度としては、０．０１～４Ｎが好ましく、１～３Ｎがより好ましい。当該酸としては、蟻酸、酢酸、塩酸、硫酸等が挙げられる。
　当該溶液のアルコール濃度としては、０～８０容量％が好ましく、０～３０容量％がより好ましい。当該アルコールとしては、メタノール、エタノール、ブタノール、イソプロパノール等が挙げられる。 For example, a partially purified product using an ion exchange resin is prepared by adjusting the pH of the culture medium to a neutral region (pH 5 to 8), adsorbing it on a strong basic ion exchange resin or a weak basic ion exchange resin, and then containing an acid. It can be obtained by elution with an aqueous alcohol solution. At this time, it is preferable to replace the ion exchange resin before adsorption with a formic acid type or an acetic acid type.
The acid concentration of the solution is preferably 0.01 to 4N, and more preferably 1 to 3N. Examples of the acid include formic acid, acetic acid, hydrochloric acid, sulfuric acid and the like.
The alcohol concentration of the solution is preferably 0 to 80% by volume, more preferably 0 to 30% by volume. Examples of the alcohol include methanol, ethanol, butanol, isopropanol and the like.
　また、例えば、多孔性合成吸着剤による部分精製物は、上記培養液のｐＨを酸性～中性領域（ｐＨ１～８）に調整後、ポリスチレン系合成吸着剤、スチレン－ジビニルベンゼン系吸着剤又はメタクリル系合成吸着剤に吸着させた後、アルコール水溶液又はケトン水溶液で溶出して得ることができる。
　当該溶液のアルコール又はケトン濃度としては、５～９９容量％が好ましく、１０～３０容量％がより好ましい。当該アルコールとしては、メタノール、エタノール、ブタノール、イソプロパノール等が挙げられる。当該ケトンとしてはアセトン等が挙げられる。 Further, for example, in a partially purified product using a porous synthetic adsorbent, after adjusting the pH of the culture solution to an acidic to neutral range (pH 1 to 8), a polystyrene synthetic adsorbent, a styrene-divinylbenzene adsorbent, or a methacrylic adsorbent is used. After adsorbing to the system synthetic adsorbent, it can be obtained by elution with an aqueous alcohol solution or an aqueous ketone solution.
The concentration of alcohol or ketone in the solution is preferably 5 to 99% by volume, more preferably 10 to 30% by volume. Examples of the alcohol include methanol, ethanol, butanol, isopropanol and the like. Examples of the ketone include acetone.
　さらに高度な精製を行うには、濃縮、沈殿化、有機溶媒による抽出、各種クロマトグラフィー等を採用することができる。 For further advanced purification, concentration, precipitation, extraction with an organic solvent, various chromatographies, and the like can be employed.
　なお、この培養液には植物成長調整作用をもつフェニル酢酸やインドール酢酸が含まれる場合がある（Kimら２００４.Current Microbiology誌４８巻３１２－３１７ページ及びWillkinsonら１９９４.Plant and Soil誌159巻２９１－２９５ページ）。その場合は、目的に応じて混合物のまま使用してもよいし、それぞれを分取して使用しても良い。
　また、培地条件によってはＮ－（フェネチル）コハク酸アミドが含まれる場合があるが、この場合も目的に応じて混合物のまま使用してもよいし、それぞれを分取して使用しても良い。 This culture solution may contain phenylacetic acid or indoleacetic acid having a plant growth regulating action (Kim et al. 2004. Current Microbiology 48, 312-317 and Willkinson et al. 1994. Plant and Soil, 159, 291). -295 pages). In that case, depending on the purpose, the mixture may be used as it is, or each of them may be collected and used.
Depending on the medium conditions, N- (phenethyl) succinamide may be contained, but in this case as well, the mixture may be used as it is depending on the purpose, or each of them may be used separately. .
　また、植物は一般にトリプトファンを生合成でき、多くの植物でトリプタミンも生合成できることが証明されている（Facchiniら２０００.Phytochemistry誌５４巻１２１－１３８ページ）。そしてそれらの一部は植物体表面、特に根面から分泌されていることが知られている（Kamilovaら２００.Molecular Plant-Microbe Interaction誌１９巻２５０－２５６ページ）。このため、これらの植物体、その種子、該植物体栽培用水又は土壌に、当該微生物株を施用又は接種すれば、植物体内乃至植物体表面でインドリルアルキルコハク酸アミドを生合成するので、植物の成長調節が可能になる。
　具体的には、接種源としては、当該菌株を通常のバチルス属微生物が増殖する条件で培養することによって得られた菌体及び／又は芽胞を含む培養液そのものや、遠心分離・膜分離などによって菌体及び／又は芽胞を濃縮したもの、あるいはさらに菌体及び／又は芽胞を水又は通常用いられる緩衝液で洗浄したものを用いることができる。これらの接種源はそのまま使用することもできるし、その乾燥物、凍結乾燥物を使用することもできる。また、ベントナイト、ゼオライト、クレーなどの無機物質担体、ピートモス、活性炭などの有機物担体に保持させて使用することもできる。
　接種する対象としては植物体全般、水、土壌に使用することができるが、当該化合物の発根促進活性が高いことを利用する場合は、種子、根、挿し木の切り口断面などが好ましい。接種濃度としては接種源１ｇあたり菌数が１×１０4～１×１０11個となるようにした上で接種すればよい。 In addition, it has been proved that plants can generally biosynthesize tryptophan, and that many plants can also biosynthesize tryptamine (Facchini et al. 2000. Phytochemistry, Vol. 54, pages 121-138). And it is known that some of them are secreted from the plant surface, especially from the root surface (Kamilova et al. 200. Molecular Plant-Microbe Interaction, Vol. 19, pages 250-256). For this reason, if these microbial strains are applied to or inoculated with these plants, their seeds, water for plant cultivation or soil, indolylalkyl succinic acid amide is biosynthesized in the plant body or on the plant body surface. Can be controlled.
Specifically, as an inoculation source, a culture solution containing bacterial cells and / or spores obtained by culturing the strain under conditions in which normal Bacillus microorganisms are grown, centrifugation, membrane separation, etc. What concentrated the microbial cell and / or spore, or what wash | cleaned the microbial cell and / or spore with water or the buffer solution normally used can be used. These inoculation sources can be used as they are, or dried or lyophilized products thereof. Further, it can be used by being supported on an inorganic substance carrier such as bentonite, zeolite, or clay, or an organic substance carrier such as peat moss or activated carbon.
The inoculation target can be used for whole plants, water and soil, but when utilizing the high rooting promoting activity of the compound, seeds, roots, cutting cross sections, etc. are preferable. The inoculation concentration may be inoculated after the number of bacteria is 1 × 10 4 to 1 × 10 11 per gram of inoculation source.
　また、本発明者は、後記実施例に示すように、一般式（３） In addition, as shown in Examples described later, the present inventor has the general formula (3)
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
（式中、Ｒ４は水素原子、ハロゲン原子、水酸基、アルコキシ基又はアルキル基を示し、ｍは０～３の数を示す）
で表されるヒドロキシインドリルアルキルコハク酸アミド化合物又はその塩が発根促進作用を有することを見出している。 (Wherein R 4 represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group or an alkyl group, and m represents a number of 0 to 3)
It has been found that a hydroxyindolylalkyl succinic acid amide compound represented by the formula:
　従って、上記一般式（３）で表される化合物又はその塩は、発根促進剤や植物成長調整剤として使用することができ、また、発根促進剤や植物成長調整剤を製造するために使用することができる。なお、「発根促進」とは、アズキ、トマト等の植物の発根を促進することをいい、「植物成長調整」は、当該植物の成長を調整することをいう。 Therefore, the compound represented by the general formula (3) or a salt thereof can be used as a rooting promoter or a plant growth regulator, and for producing a rooting promoter or a plant growth regulator. Can be used. Note that “promoting rooting” means promoting rooting of plants such as azuki bean and tomato, and “controlling plant growth” means adjusting the growth of the plant.
　上記Ｒ４、ｍの好適な具体例としては、発根促進作用の点で、それぞれＲ１、ｎと同様である。また、一般式（３）で表される化合物又はその塩としては、発根促進作用の点で、ブホブタン酸が特に好ましい。 Preferred examples of R 4 and m are the same as R 1 and n, respectively, in terms of rooting promoting action. In addition, as the compound represented by the general formula (3) or a salt thereof, bubutanoic acid is particularly preferable from the viewpoint of rooting promoting action.
　次に実施例を挙げて本発明を詳細に説明するが、本発明はこれに限定されるものではない。 Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
実施例１
　５００mL容バッフル付き三角フラスコに０質量％、０．０１質量％、０．０３質量％、０．１質量％又は０．３質量％のトリプタミンを添加したＴｒｙｐｔｉｃ　Ｓｏｙ　Ｂｒｏｔｈ（ベクトン・ディッキンソンアンドカンパニー社製、以下ＴＳＢ）を各３００mL×２本準備し、オートクレーブ滅菌後、あらかじめＴＳＢで前培養したＢａｃｉｌｌｕｓ　ｔｈｕｒｉｎｇｉｅｎｓｉｓ　ＪＣＭ２０３８６株を接種した。温度２７℃、回転数８０rpmで１０日間振とう培養した。培養温度は２７℃とし、回転数８０rpmで１０日間振とう培養した。 Example 1
Tryptic Soy Broth (manufactured by Becton Dickinson & Company) with 0%, 0.01%, 0.03%, 0.1%, or 0.3% by weight tryptamine added to a 500 mL Erlenmeyer flask with baffle The following TSB) were prepared in an amount of 2 x 300 mL, autoclaved, and inoculated with Bacillus thuringiensis JCM20386 strain that had been pre-cultured with TSB in advance. The cells were cultured with shaking at a temperature of 27 ° C. and a rotation speed of 80 rpm for 10 days. The culture temperature was 27 ° C., and shaking culture was performed at a rotation speed of 80 rpm for 10 days.
　得られた培養液は以下の通り粗精製した。培養液を６，５００rpm、３０分で遠心分離し、上澄みを塩酸でpH３．０に調整した。別途、スチレン－ジビニルベンゼン系合成吸着樹脂ダイヤイオンＨＰ－２０をメタノールで洗浄した後、カラム（内径２０mm×長さ３００mm）に充填し、pH３．０酢酸水を通液して調整した。本カラムに上記上澄み液を流すことによって、Ｎ－ ［２－（３－インドリル）エチル］コハク酸アミド（以下ＩＥＳＡ）を吸着した。カラムは１０容量％イソプロパノール５００mLで洗浄した後、３０容量％イソプロパノール５００mLでＩＥＳＡを溶出した。溶出液はエバポレータで約１００mLに濃縮し、水酸化ナトリウム水溶液を用いてpH８．０に調整して、酢酸エチルで３回抽出を行い、酢酸エチル層は棄却した。残った水相は塩酸を用いてpH２．５とし、酢酸エチル抽出を３回行った。得られた酢酸エチル層は無水硫酸マグネシウムを用いて脱水した後、溶媒を減圧下で除去した。残渣は少量の１Ｎ酢酸水で溶解し、別途メタノールで洗浄後、１Ｎ酢酸水を通液して調整したSepPak　Ｃ１８カートリッジ（Ｗａｔｅｒｓ社製）に通過させることにより、ＩＥＳＡを吸着した。カートリッジは６０容量％メタノール２０mLで溶出し、溶出液は減圧下、乾固した。 The obtained culture broth was roughly purified as follows. The culture solution was centrifuged at 6,500 rpm for 30 minutes, and the supernatant was adjusted to pH 3.0 with hydrochloric acid. Separately, styrene-divinylbenzene synthetic adsorption resin Diaion HP-20 was washed with methanol, then packed in a column (inner diameter 20 mm × length 300 mm), and adjusted by passing pH 3.0 acetic acid solution through. By flowing the supernatant liquid through this column, N- [2- (3-indolyl) ethyl] succinamide (hereinafter, IESA) was adsorbed. The column was washed with 500 mL of 10% by volume isopropanol, and IESA was eluted with 500 mL of 30% by volume isopropanol. The eluate was concentrated to about 100 mL with an evaporator, adjusted to pH 8.0 using an aqueous sodium hydroxide solution, extracted three times with ethyl acetate, and the ethyl acetate layer was discarded. The remaining aqueous phase was adjusted to pH 2.5 with hydrochloric acid and extracted three times with ethyl acetate. The obtained ethyl acetate layer was dehydrated using anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure. The residue was dissolved in a small amount of 1N aqueous acetic acid, separately washed with methanol, and passed through a SepPak C18 cartridge (manufactured by Waters) prepared by passing through 1N aqueous acetic acid to adsorb IESA. The cartridge was eluted with 20 mL of 60% by volume methanol, and the eluate was evaporated to dryness under reduced pressure.
　このサンプルはＨＰＬＣ（カラム、ＹＭＣ－Ｐａｃｋ Ｐｈｅ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間８．５～１１．５分）を分取した。分取した画分はＨＰＬＣ（カラム、ＯＤＳ-Ａ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間１１～１３分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、Ｐｏｌａｒ ＲＰ　内径１０mm×長さ２５０mm（Ｐｈｅｎｏｍｅｎｅｘ社製）；移動相、１容量％酢酸含有３０容量％アセトニトリル；流速、３．０mL／分）で分析した。ピークは１４．３分に認められ、これは化学合成したＩＥＳＡとほぼ一致した。このピークの紫外部吸光スペクトルを測定したところ、２８１nmに吸光極大が認められ、化学合成したＩＥＳＡとほぼ一致した。２８０nmで測定したピーク面積を化学合成したＩＥＳＡによって作成した検量線と比較し、培養液中から得られたＩＥＳＡ濃度を算出した。結果は表１に示した。
　この結果から、本菌株はＴＳＢのみで培養しても培養液中にＩＥＳＡを生産していたことが証明された。これはＴＳＢ中に培地原料として添加されているカゼイン由来のトリプトファンが約１質量％含まれるものに由来すると考えられる。さらに、トリプタミンの添加区ではＩＥＳＡ生産量が飛躍的に向上することも同時に証明された。 This sample was purified using HPLC (column, YMC-Pack Phe inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol by volume containing 1% acetic acid; flow rate, 3.0 mL / min) The fraction corresponding to the IESA retention time (retention time 8.5 to 11.5 minutes) was collected. The fraction collected was purified using HPLC (column, ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the IESA retention time (retention time 11 to 13 minutes) was collected. Further fractions were analyzed by HPLC (column, Polar RP inner diameter 10 mm × length 250 mm (Phenomenex); mobile phase, 30% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min). A peak was observed at 14.3 minutes, which almost coincided with chemically synthesized IESA. When the ultraviolet absorption spectrum of this peak was measured, an absorption maximum was observed at 281 nm, which almost coincided with chemically synthesized IESA. The peak area measured at 280 nm was compared with a calibration curve prepared by chemically synthesized IESA, and the IESA concentration obtained from the culture solution was calculated. The results are shown in Table 1.
From this result, it was proved that this strain produced IESA in the culture medium even when cultured with TSB alone. This is considered to be derived from the case where about 1% by mass of tryptophan derived from casein added as a medium raw material in TSB is contained. Furthermore, it was also proved that IESA production was dramatically improved in the area where tryptamine was added.
　また、上記分析時にＩＥＳＡ保持時間相当画分（保持時間１３．５－１５．５分）を分取し、５検体分を１サンプルにまとめて濃縮し、さらにＨＰＬＣ（カラム、ＸＴｅｒｒａＲＰ　内径１０mm×長さ２５０mm（Ｗａｔｅｒｓ社製）；移動相、１容量％酢酸含有２５容量％アセトニトリル；流速、３．０mL／分）で精製し、ＩＥＳＡ保持時間相当画分（保持時間１５～１９分）を単一ピークとして分取した。得られた画分は減圧下で濃縮後、五酸化二リン存在下、デシケータ内で減圧乾燥した結果、ＩＥＳＡの結晶５．８ｍｇを得た。この結晶についてグリセロールを用いた質量分析（ＭＳ－ＦＡＢ）によって分析したところ、（Ｍ＋Ｈ）+が２６１として検出され、さらにＮａＣｌを添加して分析したところ（Ｍ＋Ｎａ）+が２８３として検出され、化学合成品と一致した。さらに、1Ｈ－ＮＭＲでの帰属も化学合成品と一致した（表２）。
　これらの結果から、表１のＩＥＳＡは物理化学的な構造解析によってもＩＥＳＡであることが証明された。 At the time of the above analysis, the fraction corresponding to the IESA retention time (retention time 13.5 to 15.5 minutes) was collected, concentrated for 5 samples into one sample, and further HPLC (column, XTerraRP inner diameter 10 mm × long). 250 mm (manufactured by Waters); mobile phase, 25% acetonitrile by volume containing 1% acetic acid; flow rate, 3.0 mL / min), and fraction corresponding to IESA retention time (retention time 15-19 minutes) was single Sorted as a peak. The obtained fraction was concentrated under reduced pressure and then dried under reduced pressure in a desiccator in the presence of diphosphorus pentoxide. As a result, 5.8 mg of IESA crystals were obtained. This crystal was analyzed by mass spectrometry using glycerol (MS-FAB). As a result, (M + H) + was detected as 261, and further analyzed by adding NaCl, (M + Na) + was detected as 283. Matched the goods. Furthermore, the assignment by 1 H-NMR was also consistent with the chemically synthesized product (Table 2).
From these results, the IESA in Table 1 was proved to be IESA by physicochemical structural analysis.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
実施例２
　５００mL容バッフル付き三角フラスコにトリプトファンを１質量％添加したＮｕｔｒｉｅｎｔ　Ｂｒｏｔｈ（ベクトン・ディッキンソンアンドカンパニー社製、以下ＮＢ）を各３００mL×２本準備し、オートクレーブ滅菌後、あらかじめＮＢで前培養したＢａｃｉｌｌｕｓ　ｔｈｕｒｉｎｇｉｅｎｓｉｓＢ－３株（北海道別海町土壌由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＰ－６８９）及びＢ．　ｃｅｒｅｕｓＢ－６株（北海道別海町由来、同受領番号ＮＩＴＥ　ＡＰ－６９０）のうちいずれかを接種し、実施例１と同様に培養を行った。 Example 2
Two 500 mL Nutrient Broth (manufactured by Becton Dickinson & Company, NB) with 1% by mass tryptophan added to a 500 mL Erlenmeyer flask with baffle was prepared, and after Bacillus thuringiensis B precultured with NB after autoclaving. -3 strains (from Betsukai-cho soil in Hokkaido, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-689) and B. Cereus B-6 strain (derived from Betsukai-cho, Hokkaido, receipt number NITE AP-690) was inoculated and cultured in the same manner as in Example 1.
　なお、Ｂ－６株については、以下の手順で１６ｓ　ｒＤＮＡ配列による遺伝子同定を行った。ＮＢにて３７℃・２日間、好気培養したＢ６株培養液から遠心分離で菌体を回収し、ＰｒｅｐＭａｎ　Ｕｌｔｒａ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）にてＤＮＡを抽出した。ＤＮＡ溶液５μＬ、１６ｓ　ｒＤＮＡ内部配列より作成したプライマー８Ｆ（ＡＧＡＧＴＴＴＧＡＴＣＣＴＧＧＣＴＣ：配列番号１）及び１４９２Ｒ（ＧＴＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ：配列番号２）の１０pmol／μＬ溶液を各１μＬ、滅菌水５μＬ、Ｐｒｅｍｉｘ　Ｔａｑ（ＴａＫａｒａ）１２．５μＬを０．２mLマイクロチューブにて混和し、ＧｅｎｅＡｍｐTM ＰＣＲ　Ｓｙｓｔｅｍ９７００（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）にてＰＣＲ反応を行った。ＰＣＲは９４℃・４分反応後、９４℃・３０秒、５０℃・３０秒、７２℃・１分を３５サイクル行い、最後に７２℃・５分反応した。ＭｉｎＥｌｕｔｅ　ＰＣＲ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｋｉｔ（ＱＩＡＧＥＮ）にて増幅物を精製し、増幅断片の部分シーケンス解析をＳＩＧＭＡ　Ｇｅｎｏｓｙｓに依頼した。解析された約７００ｂｐについて、ＤＤＢＪ（ＤＮＡ　Ｄａｔａ　Ｂａｎｋ　ｏｆ　Ｊａｐａｎ）ホームページ（ｈｔｔｐ：／／ｆａｓｔａ．ｄｄｂｊ．ｎｉｇ．ａｃ．ｊｐ／ｔｏｐ－ｊ．ｈｔｍｌ）にてＦＡＳＴＡ検索を行い、相同性が最も高かった菌種と同定した。 For the B-6 strain, gene identification was performed using the 16s rDNA sequence according to the following procedure. Bacterial cells were collected from the culture solution of B6 strain aerobically cultured at 37 ° C. for 2 days with NB by centrifugation, and DNA was extracted with PrepMan Ultra (Applied Biosystems). 1 μL each of 10 μmol / μL solutions of primers 8F (AGAGTTTGATCCCTGCTCTC: SEQ ID NO: 1) and 1492R (GTTACCTTTGTACCACTACT: SEQ ID NO: 2) prepared from the internal sequence of 16s rDNA, 5 μL of DNA solution, 5 μL of sterilized water, 2.5 μL of Premix Taq (TaKara) 1 Were mixed in a 0.2 mL microtube, and a PCR reaction was performed using GeneAmp ™ PCR System 9700 (Applied Biosystems). PCR was performed at 94 ° C for 4 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute, and finally 72 ° C for 5 minutes. The amplified product was purified with MinElute PCR purification kit (QIAGEN), and the partial sequence analysis of the amplified fragment was requested to SIGMA Genosys. About 700 bp analyzed, FASTA search was performed on the DDBJ (DNA Data Bank of Japan) homepage (http://fasta.ddbj.nig.ac.jp/top-j.html), and the homology was highest. Identified as a bacterial species.
　培養液は実施例１と同様に粗精製を行った。粗精製したサンプルはＨＰＬＣ（カラム、ＹＭＣ Ｐａｃｋ Ｃ８内径２０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、６．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間２１～２８分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、ＹＭＣ Ｐａｃｋ Ｃ８内径２０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有３０容量％アセトニトリル；流速、６．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間２８～３４分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、ＹＭＣ－Ｐａｃｋ Ｐｈｅ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間９～１１分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、ＯＤＳ－Ａ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間１１～１３分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、Ｐｏｌａｒ ＲＰ　内径１０mm×長さ２５０mm（Ｐｈｅｎｏｍｅｎｅｘ社製）；移動相、１容量％酢酸含有３０容量％アセトニトリル；流速、３．０mL／分）で精製し、ＩＥＳＡ保持時間相当画分（保持時間１３～１５分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、Ｐｕｒｅｓｉｌ Ｃ１８　内径４．６mm×長さ２５０mm（Ｗａｔｅｒｓ社製）；カラム温度、４０℃；移動相、１容量％酢酸含有２５容量％アセトニトリル；流速、０．８mL／分）で分析した。ピークは１２．４分に認められ、これは化学合成したＩＥＳＡとほぼ一致した。このピークの紫外部吸光スペクトルを測定したところ、２８１nmに吸光極大が認められ、化学合成したＩＥＳＡと一致した。２８０nmで測定したピーク面積に基づいて実施例１と同様にＩＥＳＡを定量した結果、Ｂ－３株由来の培養液中で０．０３９mg／Ｌ、Ｂ-６株由来の培養液中で０．０３８mg／Ｌであることが判明した。本結果から、これらの菌株はＩＥＳＡを生産することが証明された。 The culture solution was roughly purified in the same manner as in Example 1. The crudely purified sample was purified using HPLC (column, YMC Pack C8 inner diameter 20 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 6.0 mL / min). The fraction corresponding to the IESA retention time (retention time of 21 to 28 minutes) was collected. Further fractions were collected using HPLC (column, YMC Pack C8 inner diameter 20 mm × length 250 mm (manufactured by YMC); mobile phase, 30% acetonitrile containing 1% acetic acid; flow rate, 6.0 mL / min). Purified and fractions corresponding to IESA retention time (retention time 28 to 34 minutes) were collected. Further, the fractions used were HPLC (column, YMC-Pack Phe, inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the IESA retention time (retention time 9 to 11 minutes) was collected. Further, the fractions were collected using HPLC (column, ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). Purified and fractions corresponding to IESA retention time (retention time 11 to 13 minutes) were collected. Further, the fraction collected was purified by HPLC (column, Polar® RP inner diameter 10 mm × length 250 mm (manufactured by Phenomenex); mobile phase, 30% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min) The fraction corresponding to the IESA retention time (retention time 13 to 15 minutes) was collected. Further, the fraction collected was HPLC (column, Puresil C18, inner diameter 4.6 mm × length 250 mm (manufactured by Waters); column temperature, 40 ° C .; mobile phase, 25% acetonitrile containing 1% by volume acetic acid; 8 mL / min). A peak was observed at 12.4 minutes, which almost coincided with chemically synthesized IESA. When the ultraviolet absorption spectrum of this peak was measured, an absorption maximum was observed at 281 nm, which was consistent with chemically synthesized IESA. As a result of quantifying IESA based on the peak area measured at 280 nm in the same manner as in Example 1, 0.039 mg / L in the culture solution derived from the B-3 strain and 0.038 mg in the culture solution derived from the B-6 strain. / L. These results proved that these strains produced IESA.
実施例３
　培地をコーンスチープリカー（和光純薬社製）を３容量％添加したＴＳＢとし、菌株をＢ－３株とした以外は実施例２と同様に培養、粗精製、分析を行った。
　その結果、０．１０２mg／ＬのＩＥＳＡが検出された。 Example 3
Culture, rough purification, and analysis were performed in the same manner as in Example 2 except that the medium was TSB to which 3% by volume of corn steep liquor (manufactured by Wako Pure Chemical Industries, Ltd.) was added and the strain was B-3.
As a result, 0.102 mg / L IESA was detected.
実施例４
　培地を、ＮＢ、ＮＢにトリプタミンを１０mM添加したもの、ＮＢにトリプタミンを１０mM添加とフェネチルアミンを３mM添加したもの、ＮＢにトリプタミンを１０mM添加とフェネチルアミンを１０mM添加したものとし、それ以外は実施例１と同様にそれぞれ培養、粗精製を行った。 Example 4
The culture medium is NB, NB with 10 mM tryptamine added, NB with 10 mM tryptamine added with 3 mM phenethylamine, NB with 10 mM tryptamine and 10 mM phenethylamine added, and otherwise with Example 1. Similarly, culture and crude purification were performed, respectively.
　粗精製後のサンプルはＨＰＬＣ（カラム、ＹＭＣ－Ｐａｃｋ Ｐｈｅ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ及びＮ－（フェネチル）コハク酸アミド（以下、ＰＥＳＡ）保持時間相当画分（保持時間８．５～１１分）を分取した。分取した画分はＨＰＬＣ（カラム、ＯＤＳ-Ａ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ及びＰＥＳＡ保持時間相当画分（保持時間１０．５～１４．０分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、Ｐｏｌａｒ ＲＰ　内径１０mm×長さ２５０mm（Ｐｈｅｎｏｍｅｎｅｘ社製）；移動相、１容量％酢酸含有３０容量％アセトニトリル；流速、３．０mL／分）で分析した。ＩＥＳＡのピークは１４．０分に認められ、これは化学合成したＩＥＳＡとほぼ一致した。このピークの紫外部吸光スペクトルを測定したところ、２８２nmに吸光極大が認められ、化学合成したＩＥＳＡとほぼ一致した。２８０nmで測定したピーク面積を化学合成したＩＥＳＡによって作成した検量線と比較し、培養液中から得られたＩＥＳＡ濃度を算出した。また、ＰＥＳＡのピークは１１．９分に認められ、これは化学合成したＰＥＳＡとほぼ一致した。このピークの紫外部吸光スペクトルを測定したところ、２５９nmに吸光極大が認められ、化学合成したＰＥＳＡとほぼ一致した。２６０nmで測定したピーク面積を化学合成したＰＥＳＡによって作成した検量線と比較し、培養液中から得られたＰＥＳＡ濃度を算出した。結果は表４に示した。
　この結果から、本発明の方法によればＩＥＳＡとＰＥＳＡを同時に生産させることも可能であることが証明された。また、ＩＥＳＡを優先的に生産させたい場合はフェネチルアミン添加量を少なくした方が有利であることが判明した。また、双方の基質濃度と生産物との関係は競合阻害と考えられるため、ＩＥＳＡ生合成酵素とＰＥＳＡ生合成酵素は同一である可能性が推察される。 The crudely purified sample was obtained using HPLC (column, YMC-Pack Phe inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). After purification, fractions corresponding to retention times of IESA and N- (phenethyl) succinamide (hereinafter referred to as PESA) (retention times of 8.5 to 11 minutes) were collected. The fraction collected was purified using HPLC (column, ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the retention time of IESA and PESA (retention time of 10.5 to 14.0 minutes) was collected. Further fractions were analyzed by HPLC (column, Polar RP inner diameter 10 mm × length 250 mm (Phenomenex); mobile phase, 30% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min). An IESA peak was observed at 14.0 minutes, which almost coincided with chemically synthesized IESA. When the ultraviolet absorption spectrum of this peak was measured, an absorption maximum was observed at 282 nm, which almost coincided with chemically synthesized IESA. The peak area measured at 280 nm was compared with a calibration curve prepared by chemically synthesized IESA, and the IESA concentration obtained from the culture solution was calculated. The PESA peak was observed at 11.9 minutes, which almost coincided with the chemically synthesized PESA. When the ultraviolet absorption spectrum of this peak was measured, an absorption maximum was observed at 259 nm, which almost coincided with chemically synthesized PESA. The peak area measured at 260 nm was compared with a calibration curve prepared by chemically synthesized PESA, and the concentration of PESA obtained from the culture solution was calculated. The results are shown in Table 4.
From this result, it was proved that IESA and PESA can be produced simultaneously according to the method of the present invention. In addition, it was found that it is advantageous to reduce the amount of phenethylamine added when it is desired to produce IESA preferentially. Moreover, since the relationship between both substrate concentrations and products is considered to be competitive inhibition, it is assumed that IESA biosynthesis enzyme and PESA biosynthesis enzyme may be the same.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例５
　５００mL容バッフル付き三角フラスコにトリプタミンを０．１質量％添加したＮＢを３００mL×２本準備し、オートクレーブ滅菌後、あらかじめＮＢで前培養したＢａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓＪＣＭ１４６５株を接種し、実施例１と同様に培養、粗精製を行った。粗精製後のサンプルは実施例４と同様にＨＰＬＣによる精製、分析を行った。その結果、０．０３２mg／ＬのＩＥＳＡが検出された。
　このことから、適当な濃度のトリプタミンを培地に添加すればＢ． ｓｕｂｔｉｌｉｓを用いてもＩＥＳＡを生産できることが明らかとなった。 Example 5
Prepare NB containing 0.1% by mass of tryptamine in a 500 mL Erlenmeyer flask with baffle, and inoculate with Bacillus subtilis strain JCM1465 pre-cultured with NB after autoclaving and culture as in Example 1. Crude purification was performed. The crudely purified sample was purified and analyzed by HPLC in the same manner as in Example 4. As a result, 0.032 mg / L IESA was detected.
Therefore, B. be added tryptamine appropriate concentrations in the medium It became clear that IESA could be produced using subtilis .
実施例６
　培地を、ＮＢにトリプトファンを１質量％添加したもの、ＮＢにトリプトファンを１質量％、硫酸マンガン５水和物（ＭｎＳＯ4・５Ｈ2Ｏ）を０．３質量％（マンガンイオン濃度として684ppm）添加し、水酸化ナトリウム水溶液でｐＨ６．８に調整したものとし、菌株をＢ－３株とした以外は実施例５と同様にそれぞれ培養、粗精製、分析を行った。その結果、硫酸マンガン無添加区で０．６４３mg／Ｌ、硫酸マンガン添加区で３．７０４mg／ＬのＩＥＳＡが検出された。
　このことから、マンガンイオンの添加によりＩＥＳＡ生産量は５倍以上高まったことが確認された。 Example 6
Medium containing 1% by mass of tryptophan in NB, 1% by mass of tryptophan in NB, and 0.3% by mass of manganese sulfate pentahydrate (MnSO 4 .5H 2 O) (684 ppm as manganese ion concentration) Then, it was adjusted to pH 6.8 with an aqueous sodium hydroxide solution, and cultured, roughly purified and analyzed in the same manner as in Example 5 except that the strain was changed to the B-3 strain. As a result, IESA of 0.643 mg / L was detected in the manganese sulfate-free group and 3.704 mg / L in the manganese sulfate-added group.
From this, it was confirmed that the addition of manganese ions increased IESA production by more than 5 times.
実施例７
　基本培地を食塩（ＮａＣｌ）０．５質量％、リン酸水素二カリウム（Ｋ2ＨＰＯ4）０．２５質量％、ブドウ糖（ｇｌｕｃｏｓｅ）０．２５質量％、硫酸マンガン五水和物（０．３質量％，マンガンイオン濃度として684ppm）水溶液とした。これに（１）酵母エキス（Ｙｅａｓｔ ｅｘｔｒａｃｔｓ、ベクトン・ディッキンソンアンドカンパニー社製）３容量％、（２）トリプトン（Ｔｒｙｐｔｏｎｅ、ベクトン・ディッキンソンアンドカンパニー社製）３質量％、（３）ソイトン（Ｓｏｙｔｏｎｅ、ベクトン・ディッキンソンアンドカンパニー社製）３質量％、（４）ソイトン３質量％およびグルタミン酸１質量％をそれぞれ加え、ｐＨ７．２に調整したものを培地とし、菌株をＢ－３株とした以外は実施例５と同様に培養、粗精製、分析を行った。結果は表４に示した。
　本結果から、培地にマンガンイオンを添加しておくことで、酵母エキス、トリプトン、ソイトンなど広範な窒素源からＩＥＳＡを生産できることが確認された。 Example 7
The basic medium is 0.5% by mass of sodium chloride (NaCl), 0.25% by mass of dipotassium hydrogen phosphate (K 2 HPO 4 ), 0.25% by mass of glucose (glucose), manganese sulfate pentahydrate (0.3%). (Mass%, manganese ion concentration 684 ppm). (1) Yeast extract (Yeast extracts, manufactured by Becton Dickinson & Company) 3% by volume, (2) Tryptone (Tryptone, manufactured by Becton Dickinson & Company) 3% by mass, (3) Soyton (Soytone, Becton) -Dickinson & Company, Inc.) Example 3 except that 3% by mass, (4) 3% by mass of soyton and 1% by mass of glutamic acid were added to adjust the pH to 7.2, and the strain was B-3. In the same manner as in No. 5, culture, crude purification, and analysis were performed. The results are shown in Table 4.
From this result, it was confirmed that IESA can be produced from a wide range of nitrogen sources such as yeast extract, tryptone, and soyton by adding manganese ions to the medium.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
実施例８
　（１）成分無調整牛乳（日本ミルクコミュニティ（株）製）そのもの、（２）当該牛乳にＬ－リンゴ酸（Ｌ－ｍａｌｉｃ ａｃｉｄ）を０．３質量％添加したもの、（３）上記牛乳に硫酸マンガン五水和物を０．３質量％（マンガンイオン濃度として684ppm）添加したもの、（４）上記牛乳にＬ－リンゴ酸（Ｌ－ｍａｌｉｃ ａｃｉｄ）０．３質量％と硫酸マンガン五水和物０．３質量％添加したものをそれぞれ調製し、ｐＨ７．２に調整して培地とし、菌株をＢ－３株とした以外は実施例５と同様に培養、粗精製、分析を行った。結果は表５に示した。
　本結果よりＢ-３株により牛乳そのものを培地としてもＩＥＳＡを生産することが可能であることが明らかとなった。また、その生産量はマンガンイオンやリンゴ酸を添加することによりさらに向上することが判明した。 Example 8
(1) Unadjusted milk (manufactured by Nippon Milk Community Co., Ltd.) itself, (2) 0.3% by mass of L-malic acid added to the milk, (3) Manganese sulfate pentahydrate added in an amount of 0.3% by mass (manganese ion concentration of 684 ppm), (4) 0.3% by mass of L-malic acid and manganese sulfate pentahydrate in the above milk Each was added with 0.3% by mass of the product, adjusted to pH 7.2, used as a medium, and cultured, roughly purified and analyzed in the same manner as in Example 5 except that the strain was B-3 strain. The results are shown in Table 5.
From this result, it became clear that IESA can be produced using B-3 strain even when milk itself is used as a medium. It was also found that the production amount was further improved by adding manganese ions and malic acid.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
実施例９
　（１）チーズホエイからＵＦ膜（Ultrafiltration Membrane）を用いてタンパク質を回収した際の透過液であるホエイＵＦ膜パーミエイト（雪印乳業（株）なかしべつ工場製）にＬ－リンゴ酸（Ｌ－ｍａｌｉｃ ａｃｉｄ）０．３質量％と硫酸マンガン五水和物を０．３質量％（マンガンイオン濃度として684ppm）添加したもの、（２）乳清パウダーＦＣ０６（雪印乳業（株）製）１２質量％水溶液、（３）当該乳清パウダー１２質量％水溶液にＬ－リンゴ酸０．３質量％と硫酸マンガン五水和物を０．３質量％添加したものをそれぞれ調製し、ｐＨ７．２に調整して培地とした。これにＢ－３株を接種し、実施例５と同様に培養、粗精製、分析を行った。結果は表６に示した。
　本結果から、ホエイＵＦ膜パーミエイトそのものを培地とし、マンガンイオンとＬ－リンゴ酸を添加した場合に、ＩＥＳＡを生産することができることが判明した。また、乳清パウダーといったホエイを培地とした場合、Ｂ－３株によってＩＥＳＡを生産することができるが、マンガンイオンとＬ－リンゴ酸を添加すればその生産量を飛躍的に向上させることができることが明らかとなった。 Example 9
(1) L-malic acid (L-malic acid) was added to whey UF membrane permeate (manufactured by Snow Brand Milk Products Co., Ltd., Nakashibetsu Factory), which is a permeate when protein is recovered from cheese whey using a UF membrane (Ultrafiltration Membrane). ) 0.3 mass% and manganese sulfate pentahydrate added 0.3 mass% (manganese ion concentration 684 ppm), (2) 12 mass% aqueous solution of whey powder FC06 (manufactured by Snow Brand Milk Products Co., Ltd.), (3) Preparation of each whey powder 12% by weight aqueous solution containing 0.3% by weight of L-malic acid and 0.3% by weight of manganese sulfate pentahydrate, adjusted to pH 7.2 It was. This was inoculated with the B-3 strain, and cultured, roughly purified and analyzed in the same manner as in Example 5. The results are shown in Table 6.
From these results, it was found that IESA can be produced when whey UF membrane permeate itself is used as a medium and manganese ions and L-malic acid are added. In addition, when whey such as whey powder is used as a medium, IESA can be produced by the B-3 strain. However, if manganese ions and L-malic acid are added, the production can be dramatically improved. Became clear.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
実施例１０
　タンパク質濃縮ホエイパウダーＷＰＣ３４Ｃ（ホエイ中タンパク質を膜濃縮後、スプレードライ乾燥したもの；雪印乳業（株）製）３質量％、食塩（ＮａＣｌ）０．５質量％、リン酸水素二カリウム（Ｋ2ＨＰＯ4）０．２５質量％、ブドウ糖０．２５質量％を蒸留水に溶解したものをホエイタンパク質基礎培地とした。
　（１）この基礎培地そのもの、（２）基礎培地にＬ－リンゴ酸０．３質量％を添加したもの、（３）基礎培地にＬ－リンゴ酸０．３質量％および硫酸マンガン五水和物０．３質量％（マンガンイオン濃度として684ppm）を添加したもの、（４）基礎培地にＤ－リンゴ酸０．３質量％および硫酸マンガン五水和物０．３％を添加したもの、（５）基礎培地にフマル酸０．３質量％および硫酸マンガン五水和物０．３質量％を添加したものを、それぞれ調製し、すべてｐＨ ７．２とした。これにＢ－３株を接種し、実施例５と同様に培養、粗精製、分析を行った。結果は表７に示した。
　本結果から、Ｂ－３株を用いることによって、ホエイタンパク質そのものを培地としてもＩＥＳＡを生産することができることが確認された。また、その生産量はマンガンイオンと有機酸を添加することによりさらに向上し、有機酸としてはＬ－リンゴ酸以外にもＤ－リンゴ酸、フマル酸でも効果的であることが判明した。 Example 10
Protein concentrated whey powder WPC34C (concentrated protein in whey, spray-dried; manufactured by Snow Brand Milk Products Co., Ltd.) 3% by mass, sodium chloride (NaCl) 0.5% by mass, dipotassium hydrogen phosphate (K 2 HPO) 4 ) Whey protein basal medium was prepared by dissolving 0.25% by mass and 0.25% by mass of glucose in distilled water.
(1) This basal medium itself, (2) basal medium supplemented with 0.3% by mass of L-malic acid, (3) basal medium with 0.3% by mass of L-malic acid and manganese sulfate pentahydrate 0.3 mass% (manganese ion concentration 684 ppm) added, (4) basal medium with D-malic acid 0.3 mass% and manganese sulfate pentahydrate 0.3% added, (5 ) A basal medium prepared by adding 0.3% by mass of fumaric acid and 0.3% by mass of manganese sulfate pentahydrate was prepared and adjusted to pH 7.2. This was inoculated with the B-3 strain, and cultured, roughly purified and analyzed in the same manner as in Example 5. The results are shown in Table 7.
From these results, it was confirmed that IESA can be produced by using the B-3 strain even when the whey protein itself is used as a medium. Further, the production amount was further improved by adding manganese ions and organic acids, and it was found that D-malic acid and fumaric acid are effective as organic acids in addition to L-malic acid.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
実施例１１
　（１）セミ脱塩乳清パウダーＦＣ１０（雪印乳業（株）製）１２質量％水溶液、（２）当該セミ脱塩乳清パウダー１２質量％水溶液に硫酸マンガン五水和物を０．３質量％（マンガンイオン濃度として684ppm）添加したもの、（３）上記水溶液に硫酸マンガン五水和物０．３質量％とＤ－リンゴ酸０．３質量％を添加したもの、（４）上記水溶液に硫酸マンガン五水和物０．３質量％とＬ－リンゴ酸０．３質量％を添加したもの、（５）上記水溶液に硫酸マンガン五水和物０．３質量％とフマル酸０．３質量％を添加したものを、それぞれ調製し、ｐＨ７．２に調整して培地とした。これにＢ－３株を接種し、実施例５と同様に培養、粗精製、分析を行った。結果は表８に示した。
　本結果から、Ｂ－３株を用いることによって、ホエイそのものを培地としてもＩＥＳＡを生産することができることが確認された。また、その生産量はマンガンイオンと有機酸を添加することによりさらに向上し、有機酸としてはＬ－リンゴ酸以外にもＤ－リンゴ酸、フマル酸でも効果的であることが判明した。 Example 11
(1) Semi-desalted whey powder FC10 (manufactured by Snow Brand Milk Products Co., Ltd.) 12% by mass aqueous solution, (2) Manganese sulfate pentahydrate is added to the 12% by mass semi-desalted whey powder aqueous solution in an amount of 0.3% by mass. (Manganese ion concentration 684 ppm) added, (3) manganese sulfate pentahydrate 0.3% by mass and D-malic acid 0.3% by mass added to the above aqueous solution, (4) sulfuric acid added to the above aqueous solution Manganese pentahydrate 0.3% by mass and L-malic acid 0.3% by mass added. (5) Manganese sulfate pentahydrate 0.3% by mass and fumaric acid 0.3% by mass in the above aqueous solution. Were added, adjusted to pH 7.2, and used as a medium. This was inoculated with the B-3 strain, and cultured, roughly purified and analyzed in the same manner as in Example 5. The results are shown in Table 8.
From these results, it was confirmed that IESA can be produced by using the B-3 strain even when whey itself is used as a medium. Further, the production amount was further improved by adding manganese ions and organic acids, and it was found that D-malic acid and fumaric acid are effective as organic acids in addition to L-malic acid.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
実施例１２
  セミ脱塩乳清パウダーＦＣ１０（雪印乳業（株）製）１２質量％水溶液に硫酸マンガン五水和物を０．３質量％（マンガン濃度として684ppm）添加し、ｐＨ７．２に調整した培地（実施例１１の（２）に相当）を３００mL調製し、５００mL用バッフル付き三角フラスコに入れ、オートクレーブ滅菌した。
　上記の培地に、あらかじめＮＢで前培養したBacillus thuringiensis Ｂ－２株（北海道別海町土壌由来）、同Ｂ－９株（北海道長沼町土壌由来）、同Ｂ－１０株（北海道別海町土壌由来）、同Ｂ-１８株（北海道由仁町土壌由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＢＰ－８５８）、同Ｂ-２１株（北海道由仁町土壌由来）、同Ｂ-２２株（北海道長沼町土壌由来）、同Ｂ-２４株（北海道別海町土壌由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＰ－８５７）、同Ｂ-２６株（北海道標津町土壌由来）、同Ｂ-３３株（北海道長沼町土壌由来）、同Ｂ-３６株（北海道標津町土壌由来）、同Ｂ-５９株（北海道標津町土壌由来）；B. cereus Ｂ－７株（北海道長沼町土壌由来）、同Ｂ－１５株（北海道長沼町土壌由来）、同Ｂ-１６株（北海道長沼町土壌由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＰ－８５６）；B. megaterium Ｂ－３２株（北海道由仁町土壌由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＢＰ－８５９）をそれぞれ接種し、実施例１と同様に培養した。
　なお、上記菌株は実施例２と同様の方法で遺伝子同定を行った。 Example 12
Semi-desalted whey powder FC10 (manufactured by Snow Brand Milk Products Co., Ltd.) A medium adjusted to pH 7.2 by adding 0.3% by mass of manganese sulfate pentahydrate (684 ppm as manganese concentration) to a 12% by mass aqueous solution (implemented) 300 mL of Example 11 (2)) was prepared, put into a 500 mL Erlenmeyer flask with a baffle, and sterilized by autoclave.
Bacillus thuringiensis B-2 strain (derived from Hokkaido Betsukai-cho soil), B-9 strain (derived from Naganuma-cho soil in Hokkaido), B-10 strain (derived from Betsukai-cho soil in Hokkaido) B-18 strain (from Hokkaido Yuni-cho soil, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE ABP-858), B-21 strain (from Hokkaido Yuni-cho soil), B-22 Shares (derived from Naganuma Town, Hokkaido), B-24 shares (derived from Betsukai Town, Hokkaido, (Japan) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center NITE AP-857), B-26 shares (Shibetsu Town, Hokkaido) Soil origin), B-33 strain (derived from Naganuma-cho soil in Hokkaido), B-36 strain (derived from soil in Shibetsu-cho, Hokkaido), B-59 strain (derived from soil in Shibetsu-cho, Hokkaido); B. cereus B-7 strain (Derived from Naganuma Town, Hokkaido), B-1 Ltd. (Hokkaido Naganuma-cho, derived from the soil), the B-16 share (Hokkaido Naganuma-cho, soil-derived, (Germany) Technology and Evaluation board mechanism, Patent Microorganisms Depositary receipt number NITE AP-856); B. megaterium B-32 strain (Hokkaido Yuni-cho soil origin, (Germany) product evaluation technology substrate mechanism patent microorganism deposit center receipt number NITE ABP-859) was inoculated and cultured in the same manner as in Example 1.
The above strains were identified by the same method as in Example 2.
  培養液は実施例１と同様に粗精製を行った。粗精製したサンプルはＨＰＬＣ（カラム、ＹＭＣ  ＯＤＳ－Ａ 内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＩＥＳＡ保持時間相当画分（保持時間１１．５～１４分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、Ｐｏｌａｒ ＲＰ　内径４．６mm×長さ２５０mm（Ｐｈｅｎｏｍｅｎｅｘ社製）；カラム温度、４０℃；移動相、１容量％酢酸含有２５容量％アセトニトリル；流速、０．８mL／分）で分析した。ピークは１４．４分に認められ、これは化学合成したＩＥＳＡとほぼ一致した。２８０nmで測定したピーク面積を用いて実施例１と同様にＩＥＳＡ濃度を算出した。結果は表９に示した。
  本結果から、マンガンを添加したホエイを培地として用いることによってB. thuringiensis、B. cereus、B. megateriumを用いてＩＥＳＡを生産することが出来ることが確認された。特にB. thuringiensisＢ－１８株（ＮＩＴＥ　ＡＢＰ－８５８）の生産能力が極めて高いことが明らかとなった。 The culture solution was roughly purified in the same manner as in Example 1. The crudely purified sample was purified using HPLC (column, YMC ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the IESA retention time (retention time 11.5 to 14 minutes) was collected. Further, the fraction collected was HPLC (column, Polar RP inner diameter 4.6 mm × length 250 mm (manufactured by Phenomenex)); column temperature, 40 ° C .; mobile phase, 25% acetonitrile by volume containing 1% by volume acetic acid; 8 mL / min). A peak was observed at 14.4 minutes, which almost coincided with chemically synthesized IESA. The IESA concentration was calculated in the same manner as in Example 1 using the peak area measured at 280 nm. The results are shown in Table 9.
From this result, it was confirmed that IESA can be produced using B. thuringiensis , B. cereus , B. megaterium by using whey supplemented with manganese as a medium. In particular, the production capacity of B. thuringiensis B-18 strain (NITE ABP-858) was found to be extremely high.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
実施例１３　Ｎ－［２-（５－水酸化インドール－３－イル）エチル］コハク酸アミド（Bufobutanoic acid）の生産
　まず、標品となるＮ－［２-（５－水酸化インドール－３－イル）エチル］コハク酸アミド（Bufobutanoic acid、以下ＢＢＡ）を下記の通り化学合成した。
　無水コハク酸１．５５ｇにアセトン３０mLを加え、室温にて撹拌しながら、５－水酸化トリプタミン塩酸塩（セロトニン塩酸塩、シグマアルドリッチジャパン（株）社製）３ｇとトリエチルアミン１．９７mLをアセトン５０mLに溶解したものをゆっくりと加え、さらに３０分撹拌を続けた。反応後、溶媒は減圧下で留去し、残滓を５％炭酸水素ナトリウム水溶液と酢酸エチルとで溶解し、分液ロート中で分液した。酢酸エチル相を棄却した後、水相は濃塩酸を用いてｐＨ２．５に調整し、酢酸エチルで抽出を行った。酢酸エチル相は無水硫酸マグネシウムを用いて脱水した後、溶媒を減圧下にて留去した。得られた合成物はＨＰＬＣ（カラム、ＹＭＣ－Ｐａｃｋ Ｃ８　内径２０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有５０容量％メタノール；流速、６．０mL／分）を用いて精製し、保持時間１２～１４分の溶出液を分取した。分取した画分は減圧乾固し、五酸化二燐共存下のデシケータ内で減圧乾燥し、１．１ｇのＢＢＡを得た。 Example 13 Production of N- [2- (5-hydroxyindol-3-yl) ethyl] succinic acid amide First, N- [2- (5-hydroxyindole-3- Yl) ethyl] succinamide (Bufobutanoic acid, hereinafter referred to as BBA) was chemically synthesized as follows.
Add 30 mL of acetone to 1.55 g of succinic anhydride and stir at room temperature with 3 g of 5-hydroxytryptamine hydrochloride (serotonin hydrochloride, Sigma-Aldrich Japan Co., Ltd.) and 1.97 mL of triethylamine in 50 mL of acetone. The dissolved one was added slowly and stirring was continued for another 30 minutes. After the reaction, the solvent was distilled off under reduced pressure, and the residue was dissolved in 5% aqueous sodium hydrogen carbonate solution and ethyl acetate and separated in a separatory funnel. After discarding the ethyl acetate phase, the aqueous phase was adjusted to pH 2.5 with concentrated hydrochloric acid and extracted with ethyl acetate. The ethyl acetate phase was dehydrated using anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The synthesized product was obtained using HPLC (column, YMC-Pack C8 inner diameter 20 mm × length 250 mm (manufactured by YMC); mobile phase, 50% methanol containing 1% acetic acid; flow rate, 6.0 mL / min). The eluate was purified and the retention time was 12-14 minutes. The fractions collected were dried under reduced pressure and dried under reduced pressure in a desiccator in the presence of diphosphorus pentoxide to obtain 1.1 g of BBA.
　培養による製造は以下の通り行った。ＮＢに０．１質量％の５－水酸化トリプタミン塩酸塩を添加し、ｐＨ６．８に調整した上で、５００mL容バッフル付き三角フラスコ２本に３００mLずつ分注し、オートクレーブ滅菌後、あらかじめＮＢで前培養したBacillus thuringiensis Ｂ－３株、及びB. cereus Ｂ－６株（北海道別海町由来、（独）製品評価技術基板機構特許微生物寄託センター受領番号ＮＩＴＥ　ＡＰ－６９０）をそれぞれ接種した。培養温度は３７℃とし、回転数８０rpmで１０日間振とう培養した。
  得られた培養液は実施例１と同様に遠心分離後、ダイヤイオンＨＰ－２０カラムへの吸着を行った。このカラムは１０容量％イソプロパノール５００mLでＢＢＡを溶出し、溶出液は実施例１と同様に溶媒抽出を行った。得られたサンプルは少量の２０容量％メタノールに溶解し、別途メタノールで洗浄後、２０容量％メタノールを通液して調製したSepPak ｔC18カートリッジ（Ｗａｔｅｒｓ社製）を通過させ、さらに２０容量％メタノール２０mLで溶出することにより精製した。 Production by culture was carried out as follows. Add 0.1% by mass of 5-hydroxytryptamine hydrochloride to NB and adjust to pH 6.8, then dispense 300 mL each into two 500 mL baffled Erlenmeyer flasks, sterilize by autoclave, and use NB in advance. The pre-cultured Bacillus thuringiensis B-3 strain and B. cereus B-6 strain (from Betsukai-cho, Hokkaido, (Germany) Product Evaluation Technology Board Organization Patent Microorganism Deposit Center Accession Number NITE AP-690) were inoculated. The culture temperature was 37 ° C., and shaking culture was performed at a rotation speed of 80 rpm for 10 days.
The obtained culture broth was centrifuged in the same manner as in Example 1 and then adsorbed onto a Diaion HP-20 column. This column was eluted with 500 mL of 10% by volume isopropanol, and the eluate was subjected to solvent extraction in the same manner as in Example 1. The obtained sample was dissolved in a small amount of 20% by volume methanol, separately washed with methanol, passed through a SepPak tC18 cartridge (Waters) prepared by passing 20% by volume methanol, and further 20 mL 20% by volume methanol. Purified by eluting with
　粗精製したサンプルはＨＰＬＣ（カラム、ＹＭＣ  Ｐｈｅ 内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有３０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＢＢＡ保持時間相当画分（保持時間１１～１４分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、ＹＭＣ  ＯＤＳ－Ａ 内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有３０容量％メタノール；流速、３．０mL／分）を用いて精製し、ＢＢＡ保持時間相当画分（保持時間９．５～１２．５分）を分取した。さらに分取した画分はＨＰＬＣ（カラム、ＯＤＳ－Ａ　内径１０mm×長さ２５０mm（ＹＭＣ社製）；移動相、１容量％酢酸含有２０容量％アセトニトリル；流速、３．０mL／分）で分析した。ピークは９．５分に認められ、これは化学合成したＢＢＡとほぼ一致した。また、ピークの吸光極大は２７９nmと化学合成したＢＢＡと一致しており、２３５～４００nmの吸光スペクトルも化学合成したＢＢＡとほぼ一致した。２８０nmで測定したピーク面積を用いて実施例１と同様にＢＢＡ濃度を算出した結果、Ｂ－３株は５．５１４mg／Ｌ、Ｂ－６株は４．６０９mg／ＬのＢＢＡを生産したことが明らかとなった。
　このことから、基質のインドリル基に水酸基を有していても、当該微生物によって対応するコハク酸アミド体を生産させることができることが明らかとなった。 The crudely purified sample was purified using HPLC (column, YMC Phe inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 30% methanol containing 1% acetic acid; flow rate, 3.0 mL / min) The fraction corresponding to the BBA retention time (retention time 11 to 14 minutes) was collected. Further, the fraction was collected by HPLC (column, YMC ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 30% methanol containing 1% acetic acid; flow rate, 3.0 mL / min). The fraction corresponding to the BBA retention time (retention time 9.5 to 12.5 minutes) was collected. Further, the fractions collected were analyzed by HPLC (column, ODS-A inner diameter 10 mm × length 250 mm (manufactured by YMC); mobile phase, 20% acetonitrile containing 1% by volume acetic acid; flow rate, 3.0 mL / min). . A peak was observed at 9.5 minutes, which almost coincided with chemically synthesized BBA. In addition, the absorption maximum of the peak coincided with 279 nm and chemically synthesized BBA, and the absorption spectrum of 235 to 400 nm almost coincided with chemically synthesized BBA. As a result of calculating the BBA concentration using the peak area measured at 280 nm in the same manner as in Example 1, the B-3 strain produced 5.514 mg / L and the B-6 strain produced 4.609 mg / L BBA. It became clear.
From this, it became clear that even if the indolyl group of the substrate has a hydroxyl group, the corresponding succinic acid amide can be produced by the microorganism.
  なお、ＢＢＡについて発根促進活性があることを確認するため、化学合成したＢＢＡ水溶液を調製し、塩酸を用いてpH7とし、アズキ発根促進アッセイ（Itagaki et al. 2003. Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid、 derived from a new category of root-promoting substance、 N-(phenethyl)succinamic acid analogs. Plant Soil 255:67-75.）に供した。アズキ切片は基部を72時間被検液に浸漬し、7日後に発生した不定根数を数えた。反復数は5本とした。その結果を表１０に示す。
　ＢＢＡ処理区では濃度依存的に顕著な発根数の増加が認められた。このことから、上記のＢＢＡ培養液を発根促進剤や植物成長調整剤として利用することが可能であることが確認された。 In order to confirm that BBA has root-promoting activity, a chemically synthesized BBA aqueous solution was prepared and adjusted to pH 7 with hydrochloric acid, and azuki root-promoting assay (Itagaki et al. 2003. Biological activities and structure-activity). relationship of substitution compounds of N- [2- (3-indolyl) ethyl] succinamic acid and N- [2- (1-naphthyl) ethyl] succinamic acid, derived from a new category of root-promoting substance, N- (phenethyl ) succinamic acid analogs. Plant Soil 255: 67-75.). Azuki bean slices were immersed in the test solution for 72 hours, and the number of adventitious roots generated after 7 days was counted. The number of iterations was 5. The results are shown in Table 10.
In the BBA-treated group, a marked increase in the number of roots was observed depending on the concentration. From this, it was confirmed that said BBA culture solution can be utilized as a rooting promoter or a plant growth regulator.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
実施例１４
　Bacillus thuringiensis Ｂ－１８株（受領番号ＮＩＴＥ　ＡＢＰ－８５８）をＮＢ１０mL中、２７℃、３日間培養し、培養液を１５，０００rpm、１０分遠心分離した。上澄を棄却した後、１０mLの滅菌水を加え、よく懸濁し、再度遠心分離することにより菌体の洗浄を行った。この洗浄をさらに２回繰り返したものに、滅菌水１０mLを加え、十分懸濁し、接種源とした。
　一方、トマト品種ハウス桃太郎（タキイ種苗）種子を８０容量％エタノールに1分浸漬し、ついで滅菌水で洗浄し、ついで１容量％次亜塩素酸ナトリウムに５分浸漬し、ついで滅菌水で２回洗浄することで種子消毒を行ったのち、あらかじめ滅菌したゲルライト（和光純薬社製）１質量％を含む培地表面に播種した。
　２７℃で３日間培養した後、発芽が確認された種子３粒を接種源に１０秒間浸漬した後、あらかじめゲルライト１質量％培地１００mLを外径４０mm×高さ１３０mmの植物培養試験管（旭テクノグラス社製）に入れて滅菌、固化したものに置床した（２反復）。蛍光灯による連続光下（２２００ルックス、２０℃）で１４日間栽培したのち、総根長をルートスキャナー（Ｃｏｍａｉｒ社製）で測定した。結果は表１１に示した。Ｂ－１８株の処理によって総根長が明らかに増加しているほか、目視による観察でも側根数の増加が認められた。 Example 14
Bacillus thuringiensis B-18 strain (reception number NITE ABP-858) was cultured in 10 mL of NB at 27 ° C. for 3 days, and the culture was centrifuged at 15,000 rpm for 10 minutes. After discarding the supernatant, 10 mL of sterilized water was added, suspended well, and centrifuged again to wash the cells. This washing was repeated two more times, and 10 mL of sterilized water was added and suspended sufficiently to obtain an inoculation source.
On the other hand, tomato variety House Momotaro (Takii seedling) seeds are immersed in 80% ethanol for 1 minute, then washed with sterile water, then immersed in 1% sodium hypochlorite for 5 minutes, and then twice with sterile water. After performing seed disinfection by washing, the seed was sown on the surface of a medium containing 1% by mass of pre-sterilized gellite (manufactured by Wako Pure Chemical Industries, Ltd.).
After culturing at 27 ° C. for 3 days, 3 seeds confirmed to germinate were immersed in the inoculation source for 10 seconds, and then 100 mL of gellite 1% by mass medium was preliminarily placed in a plant culture test tube (Asahi Techno) having an outer diameter of 40 mm × height of 130 mm. (Made by Glass Co., Ltd.) and placed on a sterilized and solidified product (two repetitions). After 14 days of cultivation under continuous light (2200 lux, 20 ° C.) with a fluorescent lamp, the total root length was measured with a root scanner (manufactured by Comair). The results are shown in Table 11. The total root length was clearly increased by the treatment with the B-18 strain, and an increase in the number of lateral roots was also observed by visual observation.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011

Claims (6)

  1. 　一般式（１）
    Figure JPOXMLDOC01-appb-C000005
     （式中、Ｒ1及びＲ2はそれぞれ独立して水素原子、ハロゲン原子、水酸基、アルコキシ基又はアルキル基を示し、Ｒ3は水素原子又はカルボキシル基を示し、ｎは０～３の数を示す）
    で表されるインドリルアルキルアミン類又はその塩を含有する培地中で、インドリルアルキルコハク酸アミド合成能を有するバチルス属に属する微生物を培養することを特徴とする、一般式（２）
    Figure JPOXMLDOC01-appb-C000006
     （式中、Ｒ1、Ｒ2及びｎは前記と同じ）
    で表されるインドリルアルキルコハク酸アミド化合物又はその塩の製造法。     General formula (1)
    Figure JPOXMLDOC01-appb-C000005
     (Wherein R 1 and R 2 each independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an alkoxy group or an alkyl group, R 3 represents a hydrogen atom or a carboxyl group, and n represents a number of 0 to 3) )
    A microorganism belonging to the genus Bacillus having the ability to synthesize indolylalkylsuccinic acid amide is cultured in a medium containing the indolylalkylamines or salts thereof represented by the general formula (2)
    Figure JPOXMLDOC01-appb-C000006
     (Wherein R 1 , R 2 and n are the same as above)
    The manufacturing method of the indolyl alkyl succinamide compound or its salt represented by these.
  2. 　Ｒ1及びＲ2がそれぞれ独立して水素原子又は水酸基であり、ｎが１である請求項１記載の製造法。 The process according to claim 1, wherein R 1 and R 2 are each independently a hydrogen atom or a hydroxyl group, and n is 1.
  3. 　培地中にマンガンイオン（Ｍｎ2+）を１～２０００ppm含有せしめることを特徴とする請求項１又は２記載の製造法。 The production method according to claim 1 or 2, wherein the medium contains 1 to 2000 ppm of manganese ions (Mn 2+ ).
  4. 　培地中に有機酸又はその塩を含有せしめることを特徴とする請求項１～３いずれかに記載の製造法。 4. The production method according to claim 1, wherein the medium contains an organic acid or a salt thereof.
  5. 　請求項１記載の一般式（１）で表されるインドリルアルキルアミン類又はその塩を生合成する植物体、種子、該植物体栽培用水又は土壌に、インドリルアルキルコハク酸アミド合成能を有するバチルス属に属する微生物を施用し、インドリルアルキルコハク酸アミドを合成させることを特徴とする植物成長調整方法。 A plant body, seeds, water for plant cultivation, or soil that biosynthesizes the indolylalkylamines represented by the general formula (1) according to claim 1 or a salt thereof has an ability to synthesize indolylalkylsuccinic acid amides. A plant growth control method comprising applying a microorganism belonging to the genus Bacillus to synthesize indolylalkylsuccinic acid amide.
  6. 　バチルス・チューリンゲンシス（Bacillus thuringiensis）Ｂ－１８（受領番号ＮＩＴＥ　ＡＢＰ－８５８）株、バチルス・チューリンゲンシスＢ－２４（ＮＩＴＥ ＡＰ－８５７）株、バチルス・セレウス（Bacillus cereus）Ｂ－１６（ＮＩＴＥ ＡＰ－８５６）株、又はバチルス・メガテリウム（Bacillus megaterium）Ｂ－３２（受領番号ＮＩＴＥ　ＡＢＰ－８５９）株。 Bacillus thuringiensis B-18 (reception number NITE ABP-858) strain, Bacillus thuringiensis B-24 (NITE AP-857) strain, Bacillus cereus B-16 (NITE AP- 856) strain, or Bacillus megaterium B-32 (reception number NITE ABP-859) strain.
PCT/JP2010/000089 2009-01-09 2010-01-08 Method for producing indolylalkyl succinic acid amide compound WO2010079763A1 (en)

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