JP2009095284A - Indole oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an indole oxidase comprising a single component and a gene encoding the enzyme. <P>SOLUTION: The indole oxidase is obtained by isolating a gene encoding an indole oxidase comprising a single component from metagenome library and using the transformant of a microorganism transformed by a recombinant vector including the gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein having indole oxidation activity, a gene encoding the protein, a method for producing the protein, and a method for producing an indigo-related compound using the protein.

  Indigo-related compounds are industrially known as dyes and have long been used as textile dyes. It is also used as a raw material for drugs such as food additives, pigments, antibacterial agents and antitumor agents. Indigo was originally made from natural indigo extracted from plants, but in the 19th century, a method for chemically synthesizing indigo was developed. On the other hand, dyeing methods using indigo-producing microorganisms are still used for some indigo dyeings in Japan.

Synthesis of indigo-related compounds using microbial enzymes has attracted attention as a low environmental load technology. Several genes and enzymes related to indole microbial conversion have already been reported (see Non-Patent Documents 1 to 4).
So far, the synthesis of indigo-related compounds from indole via the route shown in FIG. 1 has been reported. These reactions are known to proceed by dioxygenase or monooxygenase produced by microorganisms.

Ensley, B. D. etal., Science 1983. 222: 167-169 Keil, H. et al., J. Bacteriol. 1987. 169: 764-770 Hart, S. et al., J. Gen. Microbiol. 1992. 138: 211-216 Doukyu, N. et al., Appl. Microbiol. Biotechnol. 2002. 58: 543-546

  Microbial genes and enzymes related to indole substance conversion have already been reported, but all of them are enzymes (multicomponent enzymes) that function by combining multiple proteins (subunits). It is necessary to co-express multiple genes. However, co-expression of multiple genes requires advanced techniques and a lot of effort. For example, when a gene involved in indole oxidation forms an operon, a strong promoter upstream of the operon and a ribosome binding site (hereinafter referred to as `` RBS '') necessary for transcription into mRNA upstream of all genes. Need to be inserted). In addition, when a gene involved in indole oxidation does not form an operon, it is necessary to introduce each gene into a host using a plurality of vectors. When culturing such microorganisms, a selective medium to which a plurality of antibiotics are added must be used in order to stably maintain the vector in the host.

  An object of the present invention is to find a novel indole oxidase that does not include such a manufacturing defect, and to provide a gene encoding the enzyme to efficiently produce the indole oxidase by a genetic engineering technique. In addition, a useful indigo-related compound is produced by oxidation of indole using the enzyme.

As a result of intensive studies to solve the problem that co-expression of a plurality of genes is necessary for producing indole oxidase, the present inventors have extracted from a metagenomic library constructed by the present inventors. In the obtained DNA, a gene encoding a novel indole oxidase having activity with a single protein was found, and the gene was successfully expressed to obtain a novel indole oxidase. It has been confirmed that indigo-related compounds can be synthesized stably and efficiently, and the present invention has been completed.
That is, the present invention is as follows.
(1) A protein having indole oxidation activity consisting of the amino acid sequence shown in SEQ ID NO: 1 or consisting of a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 .
(2) A gene encoding the protein according to (1) above.
(3) A gene encoding a protein having indole oxidation activity comprising the following polynucleotide (a), (b) or (c):
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 or a base sequence complementary to the base sequence (b) one or several in the base sequence shown in SEQ ID NO: 2 or the base sequence complementary to the base sequence (C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence (SEQ ID NO: 2) 4) A vector comprising at least one gene selected from the genes described in (2) or (3) above.
(5) A transformant comprising the vector according to (4) above.
(6) A method for producing a protein having indole oxidation activity, comprising culturing the transformant according to (5) above and collecting a protein having indole oxidation activity from the obtained culture.
(7) A method for producing an indigo-related compound, comprising contacting the transformant according to (5) or the enzyme according to (1) above with an indole compound.
(8) The method according to (7) above, wherein the indigo-related compound is one or more compounds selected from the group consisting of indigo, indirubin, isatin and derivatives thereof.

The present invention provides a protein having a novel indole oxidation activity (hereinafter sometimes simply referred to as indole oxidase) and a gene encoding the protein. Since the indole oxidase of the present invention consists of a single component, when the enzyme is produced by genetic engineering techniques, the gene encoding the enzyme can regulate gene expression by a single promoter. . On the other hand, a conventional indole oxidase consisting of a plurality of subunits must be co-expressed by introducing a gene into a host using a plurality of vectors in the production thereof, and the stability of the gene in the host is increased. However, according to the present invention, the problems of the conventional method are solved, and the production of the enzyme by culturing the microorganism into which the above gene of the present invention has been introduced is more efficient and easy. This is very advantageous for the synthesis of related compounds.
The present invention is described in detail below.

1. Indole Oxidase The indole oxidase of the present invention catalyzes a reaction for converting indole into indigo or indirubin via isatin and Indoxyl 3-oxindole, and its estimated reaction path is shown in FIG.
The indole oxidase of the present invention consists of a single component, and its amino acid sequence is shown in SEQ ID NO: 1. However, as long as it has indole oxidation activity, not only this but a displacement type indole oxidase comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Is also included.

2. Indole Oxidase Gene The indole oxidase gene that led to the present invention was isolated using a metagenomic library constructed from activated sludge used for coke oven wastewater treatment.
The specific base sequence of the indole oxidase gene isolated in this way is shown in SEQ ID NO: 2, but the indole oxidase gene of the present invention is not only a polynucleotide having the base sequence shown in SEQ ID NO: 2. In addition, a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide easily obtained from amino acid sequence information derived from these nucleotide sequences is included.
These polynucleotides include a polynucleotide encoding indole oxidase consisting of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. A polynucleotide encoding a mutant indole oxidase, and further comprising a nucleotide sequence in which one or several nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2, A polynucleotide encoding a protein having indole oxidation activity is also included. The polynucleotide of the present invention means DNA or RNA.

Such a mutant gene can be prepared, for example, by performing PCR from a DNA prepared from another bacterium having indole oxidation activity, using a primer designed based on the base sequence of the gene.
In addition, mutants of the gene of the present invention having the above functions or activities can be synthesized by site-directed mutagenesis. The method for introducing a mutation into a gene is not limited, but a mutation introduction kit (for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)) using a commercially available site-directed mutagenesis method is used. Can be used.

Whether or not the recombinant protein produced by these mutant genes has enzyme activity is confirmed by measuring the function or activity of generating the reaction product of FIG. 1 by reacting the mutant protein with indole. can do. Furthermore, the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide that hybridizes with a sequence complementary to the sequence under stringent conditions and encodes a protein having each function or activity is also provided. Included in the indole oxidase gene of the invention.
Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, it refers to conditions under which DNA having high homology (70-80% or more) hybridizes. In the present invention, “stringent conditions” refers to, for example, heating in a solution of 2 × SSC, 0.1% SDS and 50% formamide at 25 ° C., and then in a solution of 0.1 × SSC, 0.1% SDS. The conditions for washing at 68 ° C. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and the same stringency can be realized by appropriately selecting these factors.

3. Recombinant Vector Containing Indole Oxidized Gene and Host The recombinant vector of the present invention can be obtained by linking the indole oxidized gene of the present invention to an appropriate vector by a method known per se or known.
The vector used in the present invention is not particularly limited as long as it can be replicated in a host, such as a plasmid, a cosmid, or a bacteriophage. Further, the host such as Escherichia coli and actinomycetes is not limited as long as the vector functions.
The direction and order of the gene to be introduced into the vector may be arbitrarily selected and sequenced as long as the gene is expressed and the expressed protein can function as an enzyme.

4). Transformant containing the vector of the present invention:
The transformant of the present invention can be obtained by introducing the above vector into a host microorganism and transforming, but the above recombinant vector can be introduced by electroporation, protoplast method, calcium chloride method (spheroplast method). Such a method may be performed by a known or publicly known method.
The host microorganism used for transformation is not particularly limited as long as it can express the gene of the present invention, and examples thereof include Escherichia coli, Pseudomonas bacteria, Ralstonia bacteria, and Rhizobium bacteria. Moreover, it is possible to produce a transformed microorganism having a high indole oxidation activity by using a combination of a vector, a host, an induction substrate that induces high expression, a promoter, an operator, an enhancer, and the like.

5. Production Method of Indole Oxidase In the present invention, the target indole oxidase can be obtained by culturing the microorganism having the gene encoding it and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, or disrupted cells. The method of culturing the microorganism of the present invention in a medium is performed according to a usual method used for culturing microorganisms. As a medium for culturing microorganisms such as actinomycetes and bacteria, any medium that contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and that can efficiently culture microorganisms can be used. Any of synthetic media may be used.

  For example, in the case of the recombinant E. coli shown above, it is cultured in a nutrient medium usually used in this research field, and if necessary, an inducer such as IPTG is added to increase the enzyme in the nutrient medium in which E. coli can grow. Expression can be performed. Under normal culture conditions, pre-cultured for 8-12 hours at 37 ° C in LB medium (1.0% peptone, 0.5% dry yeast extract, 1.0% NaCl) supplemented with antibiotics. As a seed, inoculate a new LB medium at a volume ratio of 1-5%, main culture at 37 ° C for 2-4 hours, add IPTG, and further culture at 30 ° C for 2-4 hours.

  When the protein of the target enzyme is produced after culturing, the enzyme protein is extracted by crushing the microbial cell. When the target protein is produced outside the cells, the culture solution is used as it is, or the cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, The target protein can be isolated and purified from Whether or not the target protein has been obtained can be confirmed by SDS-PAGE or the like.

6. Production method of indigo related compounds:
The indigo-related compound production method of the present invention is characterized in that it is carried out using the above-mentioned microorganism and / or enzyme. Specifically, the cultured cell or resting cell of the above-mentioned microorganism and / or This is a method of bringing the isolated enzyme into contact with an indole compound.
In the present invention, the indole compound to be decomposed is not limited, and examples thereof include indole and derivatives thereof. However, the substrate that reacts with the enzyme is not limited as long as it shows its activity.

  When cultured cells or resting cells are used, they are subjected to an indole compound oxidation reaction as a cell suspension prepared by suspending in an appropriate buffer. Various buffers can be used as the buffer, and water, a medium, or the like can be used instead of the buffer. The concentration of the bacterial suspension is preferably 1-2 at a turbidity of 600 nanometers, and can be increased or decreased as necessary. On the other hand, when the indole oxidase isolated and purified as described above is used, the indole compound is subjected to an oxidation reaction as an enzyme suspension prepared by suspending in an appropriate buffer solution, water or the like.

Conditions suitable for these oxidation reactions include, for example, increasing the degradation rate of indole compounds after allowing the indole oxidase of the present invention or a cell suspension containing the enzyme to act on the indole compounds as a substrate. It can be selected by measuring using liquid chromatography (HPLC) or the like. In this measurement, other analysis methods may be used together if necessary.
Examples of the present invention are shown below, but the present invention is not particularly limited to these examples.

Example 1
A metagenomic library was constructed by extracting the DNA of complex microorganisms from activated sludge used for coke oven wastewater treatment. The metagenomic library used Escherichia coli EPI300 strain as a host and a Fosmid vector as a vector, and clones containing DNA having an average length of about 30 kbp were obtained.
Subsequently, in order to isolate the indole oxidation gene, an LB medium containing 0.7% agar in which Bacillus subtilis was suspended was layered on an LB (Luria-bertani) agar medium in which the metagenomic library was cultured. Clones that formed a blocking circle due to inhibition of growth of B. subtilis around the metagenomic clones were selectively isolated, and fosmid DNA was extracted to determine the nucleotide sequence. DNA obtained therein from metagenome clones M103 strain becomes clear that a novel gene is Acyl-CoA Dehydrogenase gene homology was identified as the gene encoded by the gene is an indole oxidase, I Ndole - using substantially the c atabolizing p rotein, it was named icp.

Example 2
Using pM103, a vector DNA containing the icp gene isolated from the metagenomic clone M103, as a template, PCR was performed using the primer set designed as described below, and amplification of the DNA fragment encoding the icp gene was attempted. PCR reaction was performed at 94 ° C. for 10 seconds, 55 ° C. for 30 seconds, 72 ° C. for 60 seconds, 30 cycles using 10 ng of DNA fragment and a primer and EX-Taq polymerase (manufactured by Takara Bio Inc.) designed as follows. PCR was performed.

Primer set:
M103F-REC2; 5'-GGAATTCCATATGAACATCCAGAAAGAACCCTTTA (SEQ ID NO: 3)
M103R-REC2; 5'-CGGGATCCTTAGTCGCGTTCGCCCTCGTTGTAT (SEQ ID NO: 4)

  Using the pCR2.1 TOPO TA Cloning Kit (Novagen), the obtained DNA fragment is a pCR2.1 TOPO vector that specifically binds to the overhanging sequence of the DNA fragment amplified by PCR. (Novagen). The pCR2.1 TOPO vector contains ATG as the start codon, RBS upstream, and Lac promoter upstream, and by introducing the gene to be used in accordance with the start codon and reading frame, even genes from other strains can be introduced. Efficient expression can be expected. Using the constructed plasmid as a host, Escherichia coli TOP10 strain (Novagen) was transformed by electroporation.

Example 3
(1) The recombinant Escherichia coli obtained in Example 2 was inoculated into 3 ml LB medium and pre-cultured by shaking overnight at 37 ° C. Subsequently, 1 ml of this preculture was inoculated into a new 1 liter LB medium as an inoculum, and cultured overnight at 37 ° C. After culturing, the bacterial solution was placed in a 250 ml centrifuge tube and centrifuged at 6,000 rpm for 15 minutes to collect the cells. 40 ml of LB medium was added to the cells collected and pelleted to dissolve the bacteria, and 80 ml of chloroform was further added, followed by manual stirring for 10 minutes.
Thereafter, centrifugation was performed at 8,000 rpm for 15 minutes, and only the chloroform layer (lower layer) was transferred to a glass vial, and the chloroform in the glass vial was vaporized by blowing nitrogen gas.
The residue was dissolved in an amount of chloroform. The solution was amber and contained red and blue pigments.

(2) Using a silica gel column, two types of red and blue dyes in the solution obtained in (1) above were purified, and TLC analysis was performed under the following conditions.
Stationary phase column; ID 2.7 mm, length 15 cm, packed with silica gel Developing solvent; Chloroform: diethyl ether = 4: 1
TLC analysis conditions (Merck TLC plate 20 × 20 silica gel 60F ₂₅₄ )
Developing solvent; chloroform: diethyl ether = 1: 1

From the results of TLC analysis, recombinant Escherichia coli expressing the icp gene produced two types of pigments, which were identified as indigo and indirubin from the Rf value (Relative to Front) measurement.
This result shows that the indole produced by Escherichia coli in the cells was oxidized by the indole oxidase of the present invention and converted into indigo and indirubin (FIG. 1).

The estimated metabolic pathway of indole by bacteria is shown.

Claims (8)

  A protein having indole oxidation activity, comprising the amino acid sequence shown in SEQ ID NO: 1, or consisting of a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1.   A gene encoding the protein according to claim 1. A gene encoding a protein having indole oxidation activity comprising the following polynucleotide (a), (b) or (c).
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2 or a base sequence complementary to the base sequence (b) one or several in the base sequence shown in SEQ ID NO: 2 or the base sequence complementary to the base sequence (C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence complementary to the nucleotide sequence   A vector comprising at least one gene selected from the genes according to claim 2 or 3.   A transformant comprising the vector according to claim 4.   A method for producing a protein having indole oxidation activity, comprising culturing the transformant according to claim 5 and collecting a protein having indole oxidation activity from the obtained culture.   A method for producing an indigo-related compound, comprising contacting the transformant according to claim 5 or the enzyme according to claim 1 with an indole compound.   8. The method of claim 7, wherein the indigo-related compound is one or more compounds selected from the group consisting of indigo, indirubin, isatin and derivatives thereof.

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