JPH0823988A - Production of indigo by microbial process - Google Patents

Abstract

PURPOSE:To produce indigo for dark blue dye in high efficiency by growing microorganism capable of producing an alkene monoxygenase enzyme activity in a medium incorporated with indole, converting the indole to indigo by oxidation and collecting the product. CONSTITUTION:An indigo which is a dark blue dye for dyeing mainly cellulose fiber to dark blue color is produced in high efficiency by inoculating a microbial strain capable of producing an alkene monoxygenase enzyme [e.g. Nocardia corallina B-276 (FERM P-4094)] to a medium incorporated with indole, culturing at 30 deg.C for 12hr to effect the microbiological oxidation of indole to indigo by the microorganism, extracting and collecting the dye produced and accumulated in the medium by using chloroform as the extraction solvent and purifying the dye by silica gel column chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing indigo from a raw material India by a microbiological method. In particular, in a medium obtained by adding raw material India, a microorganism capable of producing an alkene monooxygenase enzyme or a microorganism capable of growing in a medium containing only an alkene as a carbon source is grown, The microorganism is microbiologically oxidized by a microorganism to be converted into indigo,
The present invention relates to a method for producing indigo, which comprises collecting indigo that accumulates in a medium.

[0002]

2. Description of the Related Art Indigo is a dye mainly used for dyeing cellulose fibers to a dark blue color. Indigo is prepared by reacting aniline and monochloroacetic acid with caustic soda to make sodium phenylglycine, and then reacting sodium phenylglycine with caustic soda, caustic potash, and sodium amide to synthesize indoxyl and air-oxidize indoxyl. It is commercially synthesized by the method. On the other hand, a method of producing indigo by a microorganism has been reported as a method replacing the chemical synthesis method, and a method of producing indigo by a microorganism that proceeds at normal temperature and pressure is expected.

A method for producing indigo by microorganisms includes, for example, naphthalene dioxygenase (BD Ensley, BJR
atzkin, TDOsslund, MJSimon, LPWackett and D.
T. Gibson, Science 222, 167 (1983)), xylene oxygenase (N. Mermod, S. Harayama and KNTimmis, Bio / Te.
chnol. 4,321 (1986)), benzenedioxygenase (S.Ir
ie, K.Shirai, S.Doi and T.Yorifuji, Agric.Biol.Che
m. 51, 1489 (1987)), toluene dioxygenase (GMSt
ephens, JMSidebotham, NHMann and H. Dalton, FEM
S Microbiol.Letters 57,295 (1989)), toluene monooxygenase (KMYen, MRKarl, LMBlatt, MJSi
mon, RBWinter, PRFausset, HSLu, AAHarcourt
and KKChen, J. Bacteriol. 173,5315 (1991)), etc.
A method for producing indigo from indole or tryptophan as a raw material using a microorganism that produces an enzyme that oxidizes aromatic hydrocarbons has been reported. However,
A method for producing indigo by a microbial method utilizing a hydrocarbon oxidase other than these aromatic hydrocarbon oxidases has not been established so far. In particular, it is desired to develop a new method for producing indigo by a microbial method, which is suitable for producing indigo with high efficiency.

[0004]

The present invention has solved the above problems, and an object of the present invention is to provide a novel method for producing indigo by a microbial method, which is suitable for producing indigo with high efficiency. To do. In particular, instead of an enzyme that oxidizes an aromatic hydrocarbon, an oxidation process by an enzyme that oxidizes a non-aromatic hydrocarbon is used, that is, a microorganism or a non-aromatic that produces an enzyme that oxidizes a non-aromatic hydrocarbon. An object of the present invention is to provide a method for efficiently producing indigo by utilizing a microorganism that assimilates a group hydrocarbon.

[0005]

Means for Solving the Problems The present inventors have conducted diligent research and as a result, converted the alkene into the corresponding epoxide by a microorganism having a biological activity of assimilating an alkene which is a non-aromatic hydrocarbon, or by oxidizing the alkene. It was found that a microorganism having the ability to produce an alkene monooxygenase enzyme, which is an enzyme that oxidizes an indole, which is a heterocyclic aromatic compound, is microbially oxidized and further converted into indigo, and thus the present invention is completed. Came to do.

That is, according to the present invention, a microorganism having an alkene monooxygenase enzyme-producing ability is grown in a medium containing indol, and the microorganism is used to
Method of producing indigo by a microbiological method comprising microbiologically oxidizing indigo to convert it to indigo, and collecting indigo accumulated in the medium, or a raw material of indigo-
In a medium containing added alkene, to grow a microorganism that can grow in a medium containing only an alkene as a carbon source, and the microorganism is used to microbiologically oxidize the indol to convert it into indigo. A method for producing indigo by a microbiological method, which comprises collecting the accumulated indigo.

The microorganism used in the present invention, which can grow in a medium containing only alkene as a carbon source, is collected from soil and grown by using a medium containing propylene as a sole carbon source. Can be separated. In particular, a microorganism that decomposes from propylene via propene oxide can be preferably used. Specifically, the genus Nocardia (genus Nocardia ) and Rhodococcous , which have already been reported as microorganisms having the ability to produce propene oxide from propylene.
(Rhodococcus), Xanthobacter (Zanthobacter
Microorganisms classified into the group consisting of the genus) and the genus Mycobacterium (Mycobacterium) are high in oxidizing ability from propylene to propene oxide, and are suitable microorganisms. As these bacteria, the following literature, CG van Ginkel, HG
J. Welten and JAM de Bont, Appl. Environ. Mic
Examples include the bacteria described in robiol. 53, 2903 (1987).

The microorganism capable of producing the alkene monooxygenase enzyme used in the present invention is a microorganism which produces the alkene monooxygenase enzyme which is an early oxidase involved in alkene metabolism. That is, it is a microorganism that produces an alkene monooxygenase enzyme that converts an alkene into a corresponding epoxide. For example, propylene can be used as an alkene and can be isolated from soil as a microorganism that produces the corresponding epoxide. Suitable microorganisms include Nocardia and Rhodococcou
Ability to convert alkenes categorized into the genus s (Rhodococcus), Xanthobacter (Zanthobacter) and Mycobacterium ( Mycobacterium ) and having a terminal carbon-carbon double bond into corresponding epoxides Can be mentioned, specifically, literature, C.
G. van Ginkel, HGJ Weltenand JAM de Bon
Examples include the bacteria described in T., Appl. Environ. Microbiol. 53, 2903 (1987). Further, a plasmid vector in which a gene DNA encoding an alkene monooxygenase enzyme derived from a microorganism having an ability to produce an alkene monooxygenase enzyme isolated from nature is incorporated into a plasmid by a known gene recombination technique. A microorganism having the ability to produce the alkene monooxygenase enzyme, which is obtained by constructing-, introducing it into a host microorganism and transforming it, can also be suitably used because it produces the alkene monooxygenase enzyme.

Furthermore, as a particularly preferred bacterium in the present invention, the present inventors have previously filed a patent application (Japanese Patent Application No. 5-1051).
Nocardia coralli disclosed in No. 71).
na (Nocardia Coralina) B-276 strain (FERM P-409
4) can be mentioned. In addition, the strain, Nocardia co
rallina B-276 strain (FERMP-
Escherichia coli obtained by transforming the gene DNA encoding the alkene monooxygenase enzyme produced by E. 4094), a plasmid vector in which the amoABCD gene is incorporated into a plasmid derived from Escherichia coli, that is, the alkene monooxygenase. An E. coli transformant having enzyme-producing ability, for example,
E. coli JM109 (pBB-1) strain (FERM BP-4250)
Etc. can also be mentioned as particularly preferable bacteria. The above-mentioned microorganism Nocardia corallina B-276 strain (FERM P-4094) has an accession number FE.
RM P-4094, E. coli JM109 (pBB-
1) The strain (FERM BP-4250) has been given the deposit number FERM BP-4250, and both have been deposited at the Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology.

In addition, a gene DN coding for an alkene monooxygenase enzyme derived from a microorganism isolated from nature and capable of producing an alkene monooxygenase enzyme.
The alkene monooxygener obtained by constructing a plasmid vector in which A is incorporated into a plasmid by a known gene recombination technique, and introducing it into a host microorganism to transform it.
Microorganisms capable of producing zezymes are not limited to those in which the above-mentioned host microorganism is Escherichia coli, but use plasmids derived from Nocardiform bacteria such as Nocardia (Nocardia) and Rhodococcous (Rhodococcus), Gene encoding the alkene monooxygenase enzyme of Escherichia coli
A transformant strain having the ability to produce the alkene monooxygenase enzyme, in which the host microorganism is a Nocardiform bacterium, into which a plasmid vector incorporating NA is introduced can also be preferably used. As a plasmid derived from the above-mentioned Nocardiform bacterium, the plasmid pNC500 already proposed by the present inventors (JP-A-5-244953).
(See Japanese Patent Laid-Open No. 6-73795) and plasmid pNC903 (see Japanese Patent Application No. 6-73795).

A gene DN coding for an alkene monooxygenase enzyme derived from a microorganism having the ability to produce an alkene monooxygenase enzyme isolated from the above-mentioned nature.
In the case of using a microorganism capable of producing the alkene monooxygenase enzyme, which is obtained by constructing a plasmid vector in which A is incorporated into a plasmid by a gene recombination technique and transforming it into a host microorganism, the alkene monooxygener As an enzyme, an alkene monooxygenase enzyme produced by Nocardia corallina B-276 strain (FERM P-4094) can be preferably used. The enzymes are amoA and amoC, which correspond to the small and large subunits of the epoxidase that adds oxygen to alkenes to form epoxides, and a reductor that transfers electrons from the coenzyme NADH to the epoxidase. -It is called amoD, which corresponds to ze (reductase), and the coupling protein that constitutes them in a complex enzyme system
It consists of four protein parts of amoB.

These amoA, amoB, amoC and amoD
In the gene DNA, each amino acid sequence is continuously coded as a cluster in the amoABCD gene shown in SEQ ID NO: 1. The amino acid sequence is shown corresponding to the codon of the amoABCD gene shown in SEQ ID NO: 1 (see the specification of Japanese Patent Application No. 5-105171).
Furthermore, a plasmid vector pDBB1 (FIG. 1) obtained by incorporating this suitable amoABCD gene into a commercially available Escherichia coli-derived plasmid pUC18 (purchased from Takara Shuzo Co., Ltd.).
The reference), E. Coli (E. coli) JM109 (pBB-1) strain (FERM
BP-4250). The plasmid vector
Transformants obtained by introducing pDBB1 into various E. coli host strains are E. coli (E. coli) JM109 (pBB-1) strain (F
Alkene monooxygener suitable as ERM BP-4250)
It can be used as a microorganism having the ability to produce enzyme.

In the present invention, either a microorganism capable of growing in a medium containing only an alkene as a carbon source or a microorganism capable of producing an alkene monooxygenase enzyme is selected and used as a raw material for indoles. In the medium obtained by adding, the microorganism is grown, and when the microorganism is microbiologically oxidized by the microorganism to convert into indigo, and the indigo is accumulated in the medium, the growth of the microorganism is favorable. It is preferably carried out under atmospheric conditions. That is, the alkene monooxygenase enzyme produced by these microorganisms or the oxidase involved in alkene metabolism can both maintain their enzymatic activity high under aerobic conditions. Furthermore,
By maintaining the medium under aerobic conditions, the indoxyl- or epoxide-type intermediate product that can be produced by microbiological oxidation from indole is easily converted to indigo by air oxidation. is there.

For the growth of the microorganism in the present invention, for example, (a) cells obtained by culturing and proliferating the microorganism in advance,
A method of growing under aerobic conditions in a medium obtained by adding the raw material, (b) the microorganism, other carbon source, nitrogen source, inorganic salts, and if necessary, a growth promoting substance. A method of cultivating and growing in an aerobic condition in a medium obtained by adding the starting material, Indol, to the nutrient medium contained can be preferably applied. For the growth of microorganisms, the pH of the medium should be
H 5-9, preferably pH 6-8, temperature 20-50
It is carried out for 1 to 6 days while maintaining the temperature in the range of 25 ° C, preferably 25 to 45 ° C. Further, during the growth, by appropriately adding a carbon source, a nitrogen source, and other components which are beneficial to the growth of the microorganism of the microorganism, the activity of the reaction between the microorganism and India can be maintained or enhanced. You can

In the above-mentioned (a) method of preliminarily cultivating and proliferating the microorganism, the medium for cultivating and proliferating first comprises sugars such as glucose, sucrose, molasses as a carbon source,
Starch hydrolyzate, cellulose hydrolyzate, and hydrocarbons such as propylene, ethylene, and butadiene having a high cell growth activity are used, and ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonia is used as a nitrogen source. Water, amino acids and other assimilable organic nitrogen compounds, inorganic salts such as potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, etc. If necessary, a medium supplemented with growth promoting substances such as vitamins, yeast extract, and cone plica is used. The culture medium is inoculated with the bacterial species of the microorganism and cultured under aerobic conditions to grow the bacterial cells. A culture medium obtained by adding the starting material, India, directly to the bacterial cell culture thus obtained, or to a suspension of bacterial cells or a cell suspension immobilized from the culture is added. Add.

On the other hand, in the above method (b) in which the microorganism is cultured and grown under aerobic conditions in a medium obtained by adding a raw material, Indol, to the nutrient medium, the above-mentioned (a) is used as the nutrient medium.
When inoculating and culturing the bacterial species of the microorganism using the medium for culture and growth used in the above method, indol is supplied to the medium to cause an oxidation reaction.

In any of the growth methods, the method of adding the starting material, Indol, to the medium may be such that the predetermined amount is added in advance at the start of the growth of the microorganism, or the Indol may be added in accordance with the time elapsed for the growth. May be appropriately added in a batch system or a continuous system. When using the batch method for adding indoles,
It can be supplied intermittently. Preferably, the indol added to the medium has an indol concentration of 1 m in the medium.
Select an amount that does not exceed M. After the growth of the microorganism is completed, indigo accumulated in the medium can be collected and purified by a means known per se, for example, a solvent (chloroform) extraction or the like. As a specific example, the volume of an extract obtained by extracting with boiling chloroform several times is reduced by evaporating and removing the solvent chloroform under a stream of an inert gas argon gas. The remaining extract can be dried with anhydrous sodium sulfate and then separated and purified using silica gel column chromatography or the like.

The present invention will be described in more detail with reference to the following examples.

[Example 1] 3 platinum loops of cells of Nocardia corallina B-276 strain (FERM P-4094)
G medium (Oxoid Labrenco Powder-10 g, Bacteriological Peptone 10 g, Glucose 10 g and Sodium Chloride 5 g with water added to make 1 L, and after adding 1N-caustic soda solution to pH 7.5 A liquid medium sterilized by heating in an autoclave at 120 ° C. for 15 minutes (100 ml) was inoculated into a 500 ml Sakaguchi flask and shaken at 30 ° C. for 96 hours to culture and proliferate the cells in advance.

The culture broth previously obtained by the culture growth was centrifuged to collect the cells. Next, 50 ml of the collected bacterial cells
Medium (K ₂ HPO ₄ 1.74g, MgSO ₄ / 7H ₂ O 1.5g, FeSO ₄・ H ₂ O
The suspension was resuspended in 0.05 g, deionized water (1 l, adjusted to pH 8.0) to prepare a bacterial suspension.

After further adding 2.5 ml of 10% glucose solution and indol (final concentration 1 mM) to the above-mentioned bacterial suspension, the mixture was grown at 30 ° C. for 12 hours while shaking and reacted with indol. Let Then, the dye produced and accumulated in the medium was extracted and fractionated with the solvent chloroform and purified by silica gel column chromatography. The TLC transfer ratio, the absorbance curve, and the mass spectrum of the purified dye were measured and compared with the results obtained using the standard indigo, and it was confirmed that the purified dye was the same as indigo. The amount of indigo obtained by preparative purification was determined by the conventional method (H. Kell, CM Saint and P.
A. Williams, J. Bcateriol. 169, 764 (1987)). As a result, the amount of indigo recovered after preparative purification was 20 mg per liter of medium.

[0021]

EXAMPLE 2 Nocardia corallina (Nocardia Kolari - Na) B-276 strain derived from alkene monooxyethylene Gena -. E transformed with peptidase gene coli (E. coli) JM109 (pBB-
1) 1 platinum loop of the strain (FERM-BP4250) was added with deionized water to 50 μg / ml ampicillin-containing LB medium (Bacto-tryptone 10 g, Bacto-east extract 5 g and sodium chloride NaCl 10 g) and added 1 l. Then, add 1N-caustic soda aqueous solution to adjust the pH to 7.2, and then in an autoclave at 120 ° C for 1
1.5 ml of a liquid medium sterilized by heating for 5 minutes) was inoculated, and the cells were cultured and proliferated at 37 ° C. overnight (about 14 hours) in advance. The obtained culture was again used in 100 ml of LB medium containing 50 μg / ml ampicillin.
Were inoculated and cultured at 37 ° C. for 2 hours. The final concentration in this solution is
IPTG (Isopropyl-β-D-thiogalactopy
(ranoside) was added, and the mixture was further cultured at 37 ° C. for 5 hours with shaking.
10 ml of the culture solution obtained by this series of culture was collected by centrifugation, and 2 ml of medium (K ₂ HPO ₄ 1.74 g, MgSO ₄ · 7H) was collected.
₂ O1.5g, FeSO ₄ · H ₂ O 0.05g, deionized water 1 l, pH 8.0
To prepare a bacterial suspension.

To the bacterial suspension, 100 μl of 10% glucose solution and indol (final concentration 1 mM) were added, and the mixture was reacted with indol at 30 ° C. for 12 hours while shaking. Then, the indigo produced and accumulated in the medium was separated and purified according to the procedure of Example 1. As a result of quantifying the amount of the purified indigo according to a conventional method, the amount of indigo recovered after the purification was 38 mg per 1 l of the medium.

[0023]

INDUSTRIAL APPLICABILITY In the method of the present invention, an alkene monooxygena, which is an enzyme that oxidizes an alkene and converts it into a corresponding epoxide, by a microorganism having a biological activity of assimilating an alkene that is a non-aromatic hydrocarbon Indigo can be efficiently produced from the indole by microbial oxidation by growing the microorganism having the enzyme-producing ability in a medium containing indole as a raw material. Further, in the method of the present invention, since the produced indigo accumulates in the medium, there is an advantage that it can be easily separated.

[0024]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 6379 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Peptide Origin: Nocardia coralina B-276 (FERM P-
4094) 910-1935 amoA 1979-2285 amoB 2300-3802 amoC 3805-4830 amoD GGA TCC CCA GGT CCC GAG CCA CCG CGG CGA TCG ACT TCC CCG TCT 45 CAT GCA CGA TCC GCA CAG CCC CCT CCC GCT TTT CCG CTT CTC TGA CAT CCC TAC CCC TTA TAG CTG ATG CCT CCA 135 CGG TCT CGG GGA ATG CCA ATA TGA CTT GGT CAG ATC GTA GGC GGC 180 AAG AAC CAT GGC TTC CTT GGC GGA CTT CAC AGG GGC CTC TCC TCG 225 AGG GCT CGT TCG GGC ATG GGT AGC ACC GTC GAG CGA ACC CGA GGA 270 GAG GCC CCG ATC CGG GGA GGC GAT CGG GAT GAA CGG GGA CGT GTC 315 GAG AAT CGA TCA GAC TTG GTG GGC AGA TTC GTG TCC GCC GGT GGG 360 CATG AAC CCG GCC ATC CAC GGG CAC TTT CGT GGC CAT CAG TGG 405 GCG GTT TCG TGA CCC CCT ATG GGC AGT TTT CCA TGG CCG CCG ACA 450 CAC AGG CCC GCA CCG ATG ACG AGG TAC TGC ACG ACC CGG CCG ACC 495 GCG CGG GCA TGG TTT GCT ACA AAT GAC AAC TTG ATC AAC GCA TCG 540 ATC ACG ACC GGC GCC ACT AAC GCC GCC GCG GCA AGC ATC GAC ATC 585 A AC ACC ATC GCG ACC GTC CGC CCT TCA CAT CGA CGA CAC CTC GGT 630 CGA TGT GTG CAT ACC GCA TGC ATC CGG CAA GAA CCC CGC GGA CTC 675 CGC AGC CGG TGC GGC TAG CCG ATT TCA CCC AAC CTG GGA CGT TGG 720 CTA AAC GTC TCG TGG ATG ACG GCG CCT GCA TGC TGC ATA GAC 765 CCG TCC GTG GCC GCA ATG TCC TGG TGA GGC ATG CGA GCG GCT TCG 810 ATT CCT CTC GGC GGG GCC AAG ACC GGC GGG CCG CGC CTG TCT AAC 855 CATC GCA GCA GGA AAT CGC AAC CGG ACC GAA ACG ATG GAA GGC 900 GTA GCG ATG ACG ACA GAG GCG ACG GTG GCC CGA CCG GTG GAG CTC 945 (amoA) Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu GAA GGT CAC CGG ACA TTC ACC TGG TTC ACG CCC GCC AGG CGA AAG 990 Glu Gly His Arg Thr Phe Thr Trp Phe Thr Pro A1a Arg Arg Lys CCG ACG GAG TAC GAG CTC TAC ACC GTG GGT CAA CAG TCC ACT CCG 1035 Pro Thr Glu Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro GAC GAG TGG CTG CAT GTG GAC TGG CCG CTG CGC TTC GAC GAC GGC 1080 Asp Glu Trp Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp G1y CGC GCC CCG TGG GAG GAG GAG TCG AGT GCG GTA CGG ACC TCG GAG 1 125 Arg Ala Pro Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu TGG TCG GCT TAC CGC GAC CCA CAC CAA CTG TGG CAG CGT CCC TAC 1170 Trp Ser Ala Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr GTC AGC ACG TGC AAC CAG GAC CAG CAG GCC CTC GCG CGG CTG GTC 1215 Val Ser Thr Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val CCC GTC CTG ACC ATG GGG TCG GCG GCG ATC ACG CCC ATC TGG TCG 1260 Pro Val Leu Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser CAG AAG ATC CTC GCC AGG TCC TAC GCC GCC TGG CCA TTC GTC GAG 1305 Gln Lys Ile Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu TAC GGG CTC TTC CTG AGC CTG GCC TAC GCC GTG CGC CAG GCC ATG l350 Tyr Gly Leu Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met TCC GAC ACG GTC CAG TTC AGC GTG GTG TTC CAG GCC GTG GAC CGC 1395 Ser Asp Thr Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg ATG CGG CTG CTC CAG GAC ATC GTC CAC CAC CTG GAC CAC CTG CAG 1440 Met Arg Leu Leu Gln Asp Ile Val His His Leu Asp His Leu Gln GAG TCG CCG GAA TTC AGC GAC GCC GGG GCC CGC GAG GCC TGG ATG 1 485 Glu Ser Pro Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met TCC GAC TCC ACC CTG GTC CCG ATC CGG GAA GTG ATC GAG CGC ATC 1530 Ser Asp Ser Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile GCC GCC AGC CAG GAC TGG GTG GAG ATC CTG GTC GCC GGC ACG CTC 1575 Ala Ala Ser Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu GTC TTC GAG CCT CTG GTC GGC CAC CTG GCG AAG GCC GAG TTG TTC 1620 Val Phe Glu Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe AGC CGC CGT GCG CCA ATG TTC GGG GAC GGG ACC CCG CCG GCG GTG 1665 Ser Arg Arg Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val CTG GCG TCG GCC CTG CTG GAC AGC GGC AGG CAC CTC GAA TCG GTC 1710 Leu Ala Ser Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val CAG GCG CTC GTC CGC CTC GTC TGC CAA GAC CCC GTC CAT GGC GAC 1755 Gln Ala Leu Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp CAG AAC CAG GCG ACT GTG CGG CGG TGG ATC GAG GAA TGG CAG CCG 1800 Gln Asn Gln Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro CGG TGC AAG GCG GCG GCC CAG TCC TTC CTG CCG ACG TTC TCC GAC 1 845 Arg Cys Lys Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp TGC GGC ATC GAC GCC AAG GAA AGC GCC AAC GCG CTG TCC CGG GCG 1890 Cys Gly Ile Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala CTG GCG AAC CAG CGG GCC GCC GTC GAG GGC GCC GGC ATC ACG GCA 1935 Leu Ala Asn Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala (amoB) M TG ACA GAC GTT AAG GAG ACC ACT GTG ACC AGC ACC CCC TCG GCC 1979 * et Thr Asp Val Lys Glu Thr Thr Val Thr Ser Thr Pro Ser Ala GCC GTG CCG GGA ACC AAG AAC CGC CGC GTT GGT ATC TCG CTG ATC 2024 Ala Val Pro Gly Thr Lys Asn Arg Arg Val Gly 1le Ser Leu Ile AGC AGC AGC GAC ACC GAG GCA GCT GTC GAG CAC ATC GCG GAG ACC 2069 Ser Ser Ser Asp Thr Glu Ala Ala Val Glu His Ile Ala Glu Thr CAG CCG GAC GCG AAG ATC GAC TTT CGG GAC TGC TTC TAC AAG ATC 2114 Gln Pro Asp Ala Lys Ile Asp Phe Arg Asp Cys Phe Tyr Lys Ile GAG CGT GAC GGG CAG CTC AGT TTC GAC ATG GCA GAG CTC AGT GAG 2159 Glu Arg Asp Gly Gln Leu Ser Phe Asp Met Ala Glu Leu Ser Glu ATC GCC GGT CGC GAC ATC GAC ACC GAC ATC TTC CTC GTG A AC ATG 2204 Ile Ala Gly Arg Asp Ile Asp Thr Asp Ile Phe Leu Val Asn Met AGC ACC TAC TAC GGC CGG ATC GTC GTC AGT GAC GGC CGG GTC GAC 2249 Ser Thr Tyr Tyr Gly Arg Ile Val Val Ser Asp Gly Arg Val Asp ATC TAC GCC GAA ATC CAG CCG GCC CGC TTC AAG GAC TGA GAG GAA 2294 Ile Tyr Ala Glu Ile Gln Pro Ala Arg Phe Lys Asp * ACA CC ATG GCA TCG AAC CCC ACC CAG CTC CAC GAG AAG TCG AAG 2338 (amoC) Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys TCC TAC GAC TGG GAC TTC ACC TCC GTC GAG CGG CGC CCC AAG TTC 2383 Ser Tyr Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe GAG ACG AAG TAC AAG ATG CCC AAG AAG GGC AAG GAC CCG TTC CGC 2428 Glu Thr Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg GTC CTG ATC CGT GAC TAC ATG AAG ATG GAA GCG GAG AAG GAC GAC 2473 Val Leu Ile Arg Asp Tyr Met Lys Pet Glu A1a Glu Lys Asp Asp CGG ACC CAT GGC TTC CTC GAC GGC GCC GTG CGG ACG CGT GAG GCC 2518 Arg Thr His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala ACC AGG ATT GAG CCG CGG TTC GCT GAG GCC ATG AAG ATC ATG GTG 2563 Thr Arg Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val CCG CAG CTG ACC AAC GCC GAG TAC CAG GCG GTG GCG GGC TGC GGA 2608 Pro Gln Leu Thr Asn Ala Glu Tyr Gln A1a Val Ala Gly Cys Gly ATG ATC ATC TCG GCC GTC GAG AAC CAG GAG CTC CGT CAG GGC TAC 2653 Met Ile Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr GCC GCT CAG ATG CTC GAT GAG GTG CGG CAC GCG CAG CTC GAG ATG 2698 Ala Ala Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met ACG CTA CGC AAC TAC TAC GCG AAG CAC TGG TGC GAT CCC TCC GGC 2743 Thr Leu Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly TTC GAC ATC GGT CAG CGC GGC CTG TAC CAG CAC CCC GCG GGG CTG 2788 Phe Asp Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu GTG TCC ATC GGC GAG TTC CAG CAC TTC AAT ACT GGT GAC CCG CTT 2833 Val Ser Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu GAC GTC ATC ATC GAT CTC AAC ATC GTG GCC GAG ACG GCG TTC ACG 2878 Asp Val Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr AAC ATC CTG CTG GTG GCC ACT CCA CAG GTC GCC GTG GCC AAC GGG 2923 Asn Ile Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly GAC AAC GCG ATG GCC AGC GTG TTC CTC TCG ATC CAG TCG GAC GAG 2968 Asp Asn Ala Met Ala Ser Val Phe Leu Ser Ile Cln Ser Asp Glu GCC AGG CAC ATG GCC AAC GGG TAC GGC TCG GTC ATG GCG CTG CTG 3013 Ala Arg His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu GAG AAC GAG GAC AAC CTC CCG CTG CTC AAC CAG TCT CTC GAT CGG 3058 Glu Asn Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg CAC TTC TGG CGT GCC CAC AAG GCC TTG GAC AAC GCG GTC GGA TCG 3103 His Phe Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp TGT TCG GAG TAT GGC GCC CGC AAG CGG CCA TGG AGC TAC AAG GCC 3148 Cys Ser Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala CAG TGG GAG GAA TGG GTC GTC GAC GAC TTC GTG GGC GGC TAC ATC 3193 Gln Trp Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile GAC CGA CTC AGC GAG TTC GGC GTT CAG GCT CCG GCC TGC CTT GGC 3238 Asp Arg Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly GCG GCC GCC GAC GAG GTC AAG TGG TCG CAC CAC ACC CTC GGT CAG 3283 Ala Ala Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln GTG CTG TCG GCG GTG TGG CCG CTG AAC TTC TGG CGC TCG GAC GCC 3328 Val Leu Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala ATG GGA CCG GCG GAC TTC GAG TGG TTC GAG AAC CAC TAC CCG GGC 3373 Met Gly Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly TGG AGC GCG GCC TAC CAG GGT TAC TGG GAG GGC TAC AAG GCG CTC 3418 Trp Ser Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu GCC GAC CCA GCA GGC GGA CGC ATC ATG CTC CAG GAG CTG CCG GGT 3463 Ala Asp Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly CTG CCG CCG ATG TGT CAG GTG TGC CAG GTG CCG TGC GTG ATG CCG 3508 Leu Pro Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro CGG CTG GAT ATG AAC GCC GCG CGG ATC ATC GAG TTC GAG GGG CAG 3553 Arg Leu Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln AAA ATC GCG CTG TGC AGC GAA CCC TGC CAG CGG ATC TTC ACC AAC 3598 Lys Ile Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn TGG CCG GAG GCG TAC CGC CAC CGC AAG CAA TAC TGG GCC CGC TAC 3643 Trp Pro Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr CAC GGA TGG GAC CTG GCG GAC GTC ATC GTT GAT CTC GGC TAC ATC 3688 His Gly Trp Asp Leu Ala Asp Val Ile Val Asp Leu Cly Tyr Ile CGC CCG GAC GGC AAG ACC CTC ATC GGC CAG CCG CTG CTC GAG ATG 3733 Arg Pro Asp Cly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met GAG CGG CTG TGG ACC ATC GAC GAC ATC CGG GCC CTT CAG TAC GAA 3778 Glu Arg Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Clu GTC AAG GAC CCG TTG CAG GAG GCG TG ATG ACG ACG ATC AAT GTG 3822 Val Lys Asp Pro Leu Gln Glu Ala * * (amoD) Met Thr Thr Ile Asn Val CAG CCC TTC TCA CAC GAG TAC TCG TGC GAG GAC GGC GAG AGC CTC 3867 Gln Pro Phe Ser His Glu Tyr Ser Cys Glu Asp G1y Glu Ser Leu CTC GAC GGC GCC CTG CGC AAC AGC CTG CTC CTG AAG TAC GGG TGC 3912 Leu Asp Gly Ala Leu Arg Asn Ser Leu Leu Leu Lys Tyr Gly Cys AAG CAC GGG GGC TGC GGG ACC TGC AAG GTC CGG CTG CTC GAC GGC 3957 Lys His Gly Gly Cys Gly Thr Cys Lys Val Arg Leu Leu Asp Gly GAC GTA GAG GAA CCC GGG TCG TCG TTC GCG CTG ACG CCG GAG GAC 4002 Asp Val Clu Glu Pro Gly Ser Ser Phe Ala Leu Thr Pro Glu Asp CGC GAG AAC GAC GTG ATC CTC GCG TGC GCC AGC GTG CCG CTG GAA 4047 Arg Glu Asn Asp Val Ile Leu Ala Cys Ala Ser Val Pro Leu Glu CCG TGC ACC ATC GAC GTC GAG CCG AGC GGC CTC ACG GAG GAG GAG 4092 Pro Cys Thr Ile Asp Val Glu Pro Ser Gly Leu Thr Clu Glu Glu TTC TTC TCG GGC GAC ACC TCG CGC GAG TTC CAG ACG GTC GTG GGC 4137 Phe Phe Ser Gly Asp Thr Ser Arg Glu Phe Gln Thr Val Val Gly GGT GTC GAG TTT CTC ACG GCG GAC ATC GCC CGG GTC CGG CTC CGG 4182 Gly Val Glu Phe Leu Thr Ala Asp Ile Ala Arg Val Arg Leu Arg CTA GAG CCG GGC GAG GAG ATC GCC TTC ACC GCC GGT CAG TTC GTC 4227 Leu Glu Pro Gly Glu Glu Ile Ala Phe Thr Ala Gly Gln Phe Val AAC GTC GAG GTG CCG GGC ACG GGT CTG CTG CGG ACC TTC TCG CTG 4272 Asn Val Clu Val Pro Gly Thr Gly Leu Leu Arg Thr Phe Ser Leu GCA AAC GCC CCT GAC GAC CCG TCA GTG GTG GAG CTG ATC TGC AAG 4317 Ala Asn Ala Pro Asp Asp Pro Ser Val Val Glu Leu Ile Cys Lys CTC TAC CCG GAT GGC CTC TTC TCC CGC TTC CTG AGG GAC GAG GCT 4362 Leu Tyr Pro Asp Gly Leu Phe Ser Arg Phe Leu Arg Asp Glu Ala GCC CCG GGC ACG CCG GTC CGG GTG TTC GGG CCG TAT GGT CAG CTC 4407 Ala Pro Gly Thr Pro Val Arg Val Phe Gly Pro Tyr Gly Gln Leu AAG ATC CGC TTG TCC CAC CGG CCG ATC CTG ATG ATC GCC GGT GGG 4452 Lys Ile Arg Leu Ser His Arg Pro Ile Leu Met Ile Ala Gly Gly TCC GGT CTC GCC CCG CTG CTC TCG ATG CTG CGA GAC TTG GCC GCC 4497 Ser Gly Leu Ala Pro Leu Leu Ser Met Leu Arg Asp Leu Ala Ala AAG AAG TGC GAC CGG CCG GTC TCG ATG TTC TTC GGC GCA CGC AGC 4542 Lys Lys Cys Asp Arg Pro Val Ser Met Phe Phe Gly Ala Arg Ser GTC GAC GAC CTG TAC CTC ATC GAG GAG ATC CGC GAG ATC GGC GAG 4587 Val Asp Asp Leu Tyr Leu Ile Glu Glu Ile Arg G1u Ile Gly Glu TCG CTA GCC GAT TTC GAG TTC ATC CCC GTG CTC TCG GAG TCG TCG 4632 Ser Leu Ala Asp Phe Glu Phe Ile Pro Val Leu Ser Glu Ser Ser CCA GCC GAC TGG CAC GGC GAG ACG GGC ATG GTC ACC GAC GCC TTG 4677 Pro Ala Asp Trp His Gly Glu Thr Gly Met Val Thr Asp Ala Leu CTG CGG TGG CGC GCC GAA CTG GCG CAT GAC GTC TAC CTG TGC GGG 4722 Leu Arg Trp Arg Ala Glu Leu Ala His Asp Val Tyr Leu Cys Gly CCG CCA CCC ATG ATC GAC GCC GCT GTG CCG CTG CTC GTC GAG CGG 4767 Pro Pro Pro Met Ile Asp Ala Ala Val Pro Leu Leu Val Glu Arg GGC GTG CGC CCA CGC AAC ATC TAC TAC GAC GCA TTC ACC CCA GCT 48l2 Gly Val Arg Pro Arg Asn Ile Tyr Tyr Asp Ala Phe Thr Pro Ala GCT CAG GTA GTC GTC GTC TGA TGG TGC ATA TCC GAT TGG GCG GCC 4857 Ala Gln Val Val Val Val Val * GGT ACC GGC GGG GTT AGG GCA GGG TAA TCG GCC GCG ATA GAG GCA 4902 GCC AGC AGA GAT GAT GCG CGT GCA AAG CGA AGC TTC GAC TCA GTT 4947 GAC ACC TTC GGC AAT GTG CTG TGC GAG CAC GAT AAC CGT GTG TTA 4992 GTG AGG CTC AAT CGC ACC GGT GCC CTG AGC GCA ATC AAT TCA 5037 GCT ATG GCT GAT CAA TTG CGG GAA GCG TGG GTG TGG GTG CGC AGC 5082 CAA ACC GGA ATT CAT TCA ATC GTC ATC TCG GCT TCG CGG CCG GAG 5127 TCG TCG CAT CGG TTT TGA TCC GGC CGA CCC GCC GGA GCC GAT 5172 GTT CGA TCG CCC CAT CAG CCC GAA GGA ATG CGG CGT CGA TCA ACG 5217 CGT CAT AGT CGC GGT GAA CGG CAT CGC GTG CCG CAT CCT GAG CGC 5262 CGG CGA A CC GAC ACT GCG TAA GCC GGT GAC GGC GGA ACA GGC GAT 5307 GGA GTC GGG CTT GGT TCG GGA GGT CGT ACC GCT TGC ATC GGC TGC 5352 GTT GTC GAG CGA ATA GCT CAG GCG GCA GGA CAT CGT ATA GGG ATT 5397 CAG GTG GGT A TGA TCT TCC GCG TGT CGA TGT GTG ACG GGC CGG 5442 ATC TGG CCT ACG AAC AAC ATC AGC AAG CGT GAC CTT CGG CCC ACA 5487 ACA TCG TGC CGT CCG CGA CCG GTG CCG ACC GTG ATC TCG GCG AAG 5532 TCC TGC CGG AGG TGT GGC GGT TCC ACA GCG TTG GGG GTC CCT 5577 CGG CTA CAC CTC GGC GCT GTT CGT CAG CTC CGA CAT CAT CGG TCA 5622 CCC ACA CTC CTG CCC GGT GTT CGG CAA CAT GGT GCA CGA CGG CCA 5667 GGT GAA CCT TCG ACA GCT AGT ACG AAT GGG CGA TGC CAA TCC 5712 GTT GCT CGA CCT CGT CGG GGT CAT GGC TGG CGT TCG CTG ACT AGG 5757 CGG CCT GTT CGT GCC GGC CGC AAC ATC CAG CCC GTG AGG AGT TGC 5802 GTA CCG GTC GGC GAG AAA CTC CAC GAC GGC AAC CTC GTC GGG 5847 CTC ACC GAG TCG GCC GAC TGG GAT CTT CGC GAT GAC CCG ATC GAG 5892 CGC CTC CTT GGG CAC CGC AGC AAC TAT CTC GGT GGA GAT GTA GCC 5937 AGG CGG AAC GGA ATT GAC GGT GAT GC C CTT GCG GGC GGT CTC CTG 5982 GGC CAG GGT CTG TGT CCT TAT CTC TTG TCG ACC GAA CAC GGT GTG 6027 CGG TCG AGT TGG TCG GTT TGA TCA GGC GGG AAC GCG GAA TAG GAG 6072 GGC TTC GCC CTG ACC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCA GCA GCC GGC CGC CCC GAC 6117 GCC GCC GCC GCG GCG GGC CAG CTC GAG CAC GAG ATG CAG CGC GAT 6162 CCG GGC GCC GGA GGC GCC GAC CGG GTG CCC CAT GGC GAT CGC GCC 6207 GCC GTT GAC GTT GAC GTT GTC GGG CAC GAT CTG GTC GCG CAT 6252 GGA TTG AAT AGC GAC GGC CGC GAA TGC CTC GTT GAT CTC GTA CAG 6297 GTC CAC ATC CAT GGC GGA GAG TCC TTC GCG GCG CAG CGC CCT GGC 6342 GAT CGC GTT GGA GGG TTG CGA CAG CAG CGA GGG ATC CGA GGG ATC

[Brief description of drawings]

[Figure 1] E. coli JM109 (pBB-1) strain (FERM
BP-4250) -containing plasmid vector pDBB1
The restriction map of and the integration site of the amoABCD gene are shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12P 17/16 C12R 1:19) (C12N 15/09 ZNA C12R 1: 365) C12R 1: 365 )

Claims (7)

[Claims] 1. A microorganism having an alkene monooxygenase enzyme-producing ability is grown in a medium containing indol, and the indol is microbiologically oxidized by the microorganism to be converted into indigo. And a method for producing indigo by a microbiological method, which comprises collecting the indigo accumulated in the medium. 2. A medium containing indol added to grow a microorganism capable of growing in a medium containing only alkene as a carbon source, and the microorganism is microbiologically oxidized to the microorganism. A method for producing indigo by a microbiological method, which comprises converting into indigo and collecting indigo accumulated in a medium. 3. The above-mentioned microorganism is a microorganism capable of producing the alkene monooxygenase enzyme, which is obtained by introducing and transforming a plasmid vector containing the gene DNA of the alkene monooxygenase enzyme. The method for producing indigo by the microbiological method according to claim 1. 4. The above-mentioned microorganisms are Nocardia (Nocardia), Rhodococcous (Rhodococcus), Xantho.
The microbiological method according to claim 1, 2 or 3, which is a microorganism classified into a genus selected from the group consisting of the genus Bacter (genus Zanthobacter) and the genus Mycobacterium (genus Mycobacterium ). Manufacturing method of indigo. 5. The above-mentioned microorganism is Nocardia corallin
a (Nocardia Coralina) B-276 strain (FERM P-4094)
The method for producing indigo by the microbiological method according to claim 1, 2 or 4, wherein 6. The alkene monooxygenase enzyme gene DNA derived from Nocardia corallina B-276 strain (FERM P-4094). The method for producing indigo by the microbiological method according to claim 3 or 4, wherein 7. The microorganism according to claim 3, wherein the alkene monooxygenase enzyme-producing ability is a transformed microorganism classified into Eschericha coli (E. coli). A method for producing indigo by a microbiological method.