JP5789366B2 - Novel styrene monooxygenase, method for producing the same, and method for producing optically active styrene oxide using the same - Google Patents

Description

  The present invention relates to a DNA encoding a novel styrene monooxygenase, a protein encoded by the DNA, and the like. The present invention also relates to a method for producing optically active styrene oxide using the protein, a method for decomposing styrene, and the like.

  Styrene is an important chemical substance in the field of chemical industries such as synthetic polymers, and is used in large quantities as a raw material for many synthetic resins such as polystyrene and styrene butadiene rubber. Gaseous styrene and styrene-containing water emissions generated in the synthesis of such synthetic resins are often problematic due to toxicity and malodor to humans.

In order to evaluate the effect of styrene on living organisms, the mechanism of styrene metabolism in mammals has been studied (Non-Patent Documents 1 to 3). In mammals, styrene is oxidized to styrene oxide by cytochrome P-450 monooxygenase [EC 1.14.14.1] (Non-patent Document 4). The styrene oxide is then detoxified by several routes. The main pathway proceeds through the formation of styrene glycol by the addition of water molecules. The main metabolites of styrene glycol are mandelic acid, phenylglyoxylic acid, and hippuric acid (Non-patent Document 5). Several styrolytic bacteria such as Pseudomonas sp., Xanthobacter sp., And Rhodococcus sp. Have been isolated from soil and their metabolic genes have been characterized (non- Patent Documents 6 to 10).
Two different pathways have been reported for the degradation of styrene by bacteria. The first pathway is a pathway involving the oxidation of vinyl side chains. The second route is a route including oxidation of an aromatic ring (Non-patent Documents 11 and 12).

In previous studies, the present inventors reported the styrene-utilizing bacteria Rhodococcus sp. ST-5 and ST-10 strains isolated from soil. These bacteria have an enzyme activity involved in styrene metabolism (Non-patent Document 13). Furthermore, the present inventors proposed the degradation pathway of styrene in the ST-5 and ST-10 strains as follows.
Styrene → styrene oxide → phenylacetaldehyde → 2-phenylethanol and phenylacetic acid.
However, genes involved in styrene degradation have not been found from these strains, and details of the metabolic mechanism remain unclear.

Korn, M., R. Wodarz, W. Schoknecht, H. Weichardt, and E. Bayer. 1984. Styrene metabolism in man: gas chromatographic separation of mandelic acid enantiomers in the urine of exposed persons. Arch. Toxicol. 55:59 -63. Lof, A., E. Gullstrand, E. Lundgren, and MB Nordqvist. 1984. Occurrence of styrene-7,8-oxide and styrene glycol in mouse after the administration of styrene. Scand. J. Work Environ. Health 10: 179 -187. Watabe, T., M. Isobe, T. Sawahata, K. Yoshikawa, S. Yamada, and E. Takabatake. 1978. Metabolism and mutagenicity of styrene. Scand. J. Work Environ. Health 4 (Suppl): 142-155 . Vainio, H., H. Norppa, K. Hemminki, and M. Sorsa. 1981. Metabolism and genotoxicity of styrene. Adv. Exp. Med. Biol. 136: 257-274. Sumner, S. C. J., and T. R. Fennell. 1994. Review of the metabolic fate of styrene. Crit. Rev. Toxicol. 24 (Suppl): S11-33. Beltrametti, F., AM Marconi, G. Bestetti, C. Colombo, E. Galli, M. Ruzzi, and E. Zennaro. 1997. Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Appl. Environ. Microbiol, 63: 2232-2239. Hartmans, S., M. J. van der Werf, and J. A. de Bont. 1990. Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase. Appl. Environ. Microbiol. 56: 1347-1351. Panke, S., B. Witholt, A. Schmid, and MG Wubbolts. 1998. Towards a biocatalyst for (S) -styrene oxide productio: characterization of the styrene degradation pathway of Pseudomonas sp. Strain VLB120. Appl. Environ. Microbiol. 64: 2032-2043. Velasco, A., S. Alonso, J. L. Garcia, J. Perera, and E. Diaz. 1998. Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. Strain Y2. J. Bacteriol. 180: 1063-1071. Warhurst, A.M., K. F. Clarke, R. A. Hill, R. A. Holt, and C. A. Fewson. 1994. Metabolism of styrene by Rhodococcus rhodochrous NCIMB 13259. Appl. Environ. Microbiol. 60: 1137-1145. Mooney, A., P. G. Ward, and K. E. O'Connor. 2006. Microbial degradation of styrene: biochemistry, molecular genetics, and perspectives for biotechnological applications. Appl. Microbiol. Biotechnol. 72: 1-10. Warhurst, A. M., and C. A. Fewson. 1994. Microbial metabolism and biotransformations of styrene. J. Appl. Bacteriol. 77: 597-606. Itoh, N., K. Yoshida, and K. Okada. 1996. Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism. Biosci. Biotech. Biochem. 60: 1826-1830.

  An object of the present invention is to provide a novel DNA encoding a styrene monooxygenase having an activity of oxidizing a vinyl group of styrene to an oxiranyl group. The present invention also provides a protein encoded by the DNA, a method for producing the protein, and the like. Furthermore, an object of the present invention is to provide a method for decomposing styrene and a method for producing optically active styrene oxide using a DNA encoding styrene monooxygenase or a transformed cell transformed with a vector containing the DNA. .

In order to isolate the styrene metabolism gene involved in the degradation of styrene in Rhodococcus sp. ST-5 and ST-10 strains, the present inventors have used the genomic DNA of ST-5 and ST-10 strains. Degenerate PCR and TAIL-PCR (thermal asymmetric interlaced PCR) were performed. As a result, the present inventors succeeded in finding four open reading frames (ORF) in the ST-5 strain. The deduced amino acid sequences encoded by these four ORFs showed similarity to the styrene metabolism gene styABCD in Pseudomonas sp. Therefore, the four ORFs were determined to be genes corresponding to styrene monooxygenase (SMO), flavin oxidoreductase, styrene oxide isomerase (SOI), and phenylacetaldehyde dehydrogenase (PAD), respectively (FIGS. 1 and 2). .
On the other hand, for the ST-10 strain, we succeeded in finding three ORFs. Two of these ORFs were determined to be genes corresponding to SMO and flavin oxidoreductase (FIGS. 1 and 2). The remaining one showed a high similarity to a protein of unknown function in which a homolog is found only in Rhodococcus.
The present inventors have completed the present invention by clarifying the action of polypeptides encoded by these genes. That is, the present invention relates to a DNA encoding a novel styrene monooxygenase, a styrene monooxygenase encoded by the DNA, a method for producing the styrene monooxygenase, a method for decomposing styrene using the styrene monooxygenase, and an optically active styrene oxide Related to the manufacturing method.

More specifically, the present invention relates to the following [1] to [9].
[1] The polynucleotide according to any one of (a) to (e) below:
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 or 3;
(B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(C) the amino acid sequence of SEQ ID NO: 2 or 4, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and described in (1) and (2) below A polynucleotide encoding a styrene monooxygenase having the following physicochemical properties:
(D) hybridizes to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 under highly stringent conditions, and has the physicochemical properties described in (1) and (2) below: A polynucleotide encoding styrene monooxygenase having; and (e) a physicochemical compound described in (1) and (2) below having at least 70% identity with the amino acid sequence set forth in SEQ ID NO: 2 or 4 A polynucleotide encoding a styrene monooxygenase having properties;
(1) Action
FADH ₂ (reduced FAD) is used as a coenzyme to act on styrene and 3-chlorostyrene to produce optically active styrene oxide and optically active 3-chlorostyrene oxide, respectively.
(2) Molecular weight This is a monomer enzyme having a molecular weight of 59,000 including histidine in gel filtration, a molecular weight of 49,000 by SDS-PAGE, and an estimated molecular weight of 47,000 from the amino acid composition.
[2] The polynucleotide according to [1], which is derived from a microorganism belonging to the genus Rhodococcus.
[3] The polynucleotide according to [2], wherein the microorganism belonging to the genus Rhodococcus is Rhodococcus opacus ST-5 strain or Rhodococcus erythropolis ST-10 strain.
[4] A protein encoded by the polynucleotide according to any one of [1] to [3].
[5] A vector comprising the polynucleotide according to any one of [1] to [3].
[6] A transformed cell into which the polynucleotide according to any one of [1] to [3] or the vector according to [5] has been introduced.
[7] The method according to any one of [1] to [3], comprising a step of culturing the transformed cell according to [6] and recovering the protein encoded by the polynucleotide according to [1] from the culture. A method for producing a protein encoded by the polynucleotide described in 1.
[8] An optically active styrene oxide and / or an optically active styrene analog comprising a step of allowing the protein according to [4] to act on styrene and / or an analog thereof, and recovering a produced compound Production method of oxide.
[9] A method for decomposing styrene and / or an analog thereof, comprising the step of allowing the protein according to [4] to act on styrene and / or an analog thereof.

The present inventors have provided DNA encoding a novel styrene monooxygenase, styrene monooxygenase encoded by the DNA, and the like.
Styrene is an important substance in the chemical industry and is indispensable as a raw material for synthetic resins such as polystyrene and styrene butadiene rubber. However, styrene gas and styrene-containing wastewater generated in the production of these synthetic polymers are often problematic from the viewpoint of human toxicity and environmental pollution. The styrene monooxygenase of the present invention is useful for the decomposition and removal of styrene that causes such problems.
Further, by using the styrene monooxygenase of the present invention, optically active styrene oxide required in various fields such as pharmaceuticals, agricultural chemicals and electronic materials can be produced efficiently and economically. Thus, the styrene monooxygenase of the present invention is also useful in the production of styrene oxide.

It is a figure which shows the comparison of the styrene catabolic gene in Rhodococcus sp.ST-5 strain | stump | stock, ST-10 strain | stump | stock, and Pseudomonas sp.Y2. Arrows indicate the direction of gene transcription. Restriction enzyme sites were indicated as follows. B, Bgl II; E, Eco RI; H, Hind III; K, Kpn I; P, Pst I; Sa, Sal I; Sp, Sph I; X, Xho I. It is a figure which shows the nucleotide sequence and deduced amino acid sequence of a styrene catabolic gene of Rhodococcus sp. ST-5 strain (A) and ST-10 strain (B). A small arrow indicates the direction of transcription of the gene. The putative ribosome binding site is shown in bold. Underlined stem loop sequences are putative transcription termination signals. It is a figure which shows the HPLC analysis result of the product of the stationary cell reaction by Rhodococcus sp.ST-10 strain | stump | stock in presence of styrene (A) or presence of styrene oxide (C). (B) is a control of (C) and shows the results of HPLC analysis in the absence of cells of ST-10 strain. STY represents styrene, SO represents styrene oxide, ACP represents acetophenone, PA represents phenylacetaldehyde, SG represents styrene glycol, and PAA represents phenylacetic acid. It is a figure which shows the HPLC chromatogram result of the reaction product of phenylacetyl-CoA ligase in ST-5 strain or ST-10 strain. The control was substrate incubated without crude cell extract. PAA represents phenylacetic acid, and PA-CoA represents phenylacetyl-CoA. It is a figure which shows the styrene catabolism pathway by Rhodococcus sp.ST-5 strain and ST-10 strain. The solid line shows the enzyme reaction. The dotted line indicates natural transformation. FIG. 2 is a view showing plasmids constructed to express styA and styAB derived from both Rhodococcus sp. ST-5 strain and ST-10 strain.

Styrene monooxygenase The styrene monooxygenase of the present invention is a protein having the following physicochemical properties (1) and (2).
(1) Action
FADH ₂ (reduced FAD) is used as a coenzyme to act on styrene and 3-chlorostyrene to produce optically active styrene oxide and optically active 3-chlorostyrene oxide, respectively.
(2) Molecular weight This is a monomer enzyme having a molecular weight of 59,000 including histidine in gel filtration, a molecular weight of 49,000 by SDS-PAGE, and an estimated molecular weight of 47,000 from the amino acid composition.
The molecular weight in the gel filtration column is a molecular weight containing a histidine tag. The molecular weight in a state where a histidine tag is added, which is deduced from the amino acid sequence, is 49.6 kDa. From these, it is estimated that the styrene monooxygenase of this application is a monomer. The method for analyzing SMO molecular weight by SDS-PAGE and the method for analyzing SMO molecular weight in gel filtration are shown in the Examples.

Styrene is an aromatic hydrocarbon represented by the following chemical formula. For example, it can be produced by dehydrogenating ethylbenzene with an iron catalyst or the like.

The activity of the styrene monooxygenase of the present invention can be measured, for example, by the following procedure.
Add purified enzyme, substrate, and coenzyme of ST-5 and ST-10-derived SMO to 50 mM MES buffer at the following concentrations to make a reaction solution. Next, this reaction solution is incubated at 30 ° C. and 2,500 rpm for 30 minutes. After completion of the reaction, add 500 μl of ethyl acetate to the reaction solution and vortex for 5 minutes. Centrifuge for 5 minutes at 15,000 rpm and transfer the separated ethyl acetate phase to a new tube. Add 500 μl of ethyl acetate again to the aqueous phase, vortex for 5 minutes, and collect the ethyl acetate phase by centrifugation again. Add an appropriate amount of sodium sulfate to a total of 1000 μl of the collected ethyl acetate phase, and vortex for 2 minutes to dehydrate. The extracted sample subjected to the dehydration treatment can be analyzed for styrene monooxygenase activity using a Hewllet Packerd HP6890 gas chromatography system.

The reaction solution can have the following composition, for example.
50 mM MES Buffer (pH 7.0)
20 mM sodium formate
100μM NADH
200μM FAD
1 U formate dehydrogenase
10 U catalase
5 mM substrate (react with 10 mM substrate during substrate conversion reaction)
StyA 150μl (In the substrate conversion reaction, use the cell disruption supernatant of StyAB co-expressing cells)
StyB 100μl
total 1000μl

The analysis by gas chromatography can be performed as follows, for example.
A 1 μl sample extracted with ethyl acetate is injected into a Varian CP-Cyclodextrin-b-2,3,6-M-19-25 capillary column (25 m, 0.25 mm ID). Carrier gas (99.999% high purity helium gas) flow rate can be 2.8 ml / min, split ratio 50, linear flow rate 46 cm / s. Detection can be performed using an FID (hydrogen flame ionization detector). The vaporization chamber temperature can be 250 ° C and the detector temperature can be 250 ° C. Identification and quantification of each substance can be obtained by analyzing a sample prepared with each standard compound at a concentration of 1 mM, 5 mM, and 10 mM under the same conditions, and creating a calibration curve from the detection time and peak area. .

Enzyme activity 1 U can be defined as the enzyme activity that converts 1 μmol of substrate into product per minute.
The enzyme activity of the styrene monooxygenase of the present invention is preferably 1 U / mg or more, more preferably 5 U / mg or more, and particularly preferably 10 U / mg or more.

Styrene monooxygenase having the above physicochemical properties can be obtained by purification from a culture of Rhodococcus sp. Examples of Rhodococcus sp. Include, but are not limited to, Rhodococcus sp. ST-5 and ST-10. These microorganisms were once identified as Corynebacterium sp. ST-5 and ST-10 strains. More recently, however, the ST-10 strain has been re-identified as Rhodococcus sp. ST-10. Rhodococcus sp. ST-5 and ST-10 can be obtained as follows.
Add sterilized water to the soil samples collected at various locations and vortex. The suspension was treated with mineral salt medium (0.3 (w / v)% (NH ₄ ) ₂ SO ₄ , 0.3% KH ₂ PO ₄ , 0.1% NaCl, 0.02% MgSO ₄ 7H ₂ O, and 1.5% agar, pH 7.0. And spread in a styrene gas atmosphere. For example, the cells are cultured at 30 ° C. for 3 to 7 days, and the grown colonies are inoculated into 4 ml of LB medium and cultured. For example, 10 μl of the obtained culture broth is spread again on an inorganic salt medium and cultured at 30 ° C. in a styrene gas atmosphere. The grown colonies can be cultured again in LB medium and stocked as styrene-utilizing bacteria.

Rhodococcus sp. ST-5 and ST-10 can be cultured in a common medium used for fungal culture, such as PY medium (0.5% yeast extract, 0.5% bactopeptone, pH 7.0). it can.
Cells cultured using these media are collected, and an inorganic salt medium (0.3 (w / v)% (NH ₄ ) ₂ SO ₄ , 0.3% KH ₂ PO ₄ , 0.1% NaCl, 0.02% MgSO ₄ 7H ₂ is collected. O, and 1.5% agar, pH 7.0) again. By growing and collecting the cells under a styrene gas atmosphere, cells having high SMO activity can be obtained.
Usually, it is desirable to adjust the pH at the start of culture to 2 to 9, preferably 4 to 7, and to culture under a temperature condition of 15 to 40 ° C, preferably 20 to 35 ° C. The culture time is usually 1 to 7 days, preferably 1 to 3 days.

  The styrene monooxygenase of the present invention can be obtained by preparing a cell-free extract from the cultured cells and further purifying the cell-free extract.

  Specifically, from the microbial cells obtained as described above, a reducing agent such as 2-mercaptoethanol, phenylmethanesulfonyl fluoride (PMFS), ethylenediaminetetraacetic acid (hereinafter, Abbreviated as EDTA), crushing by using a physical impact with glass beads in a buffer solution containing protease inhibitors such as pepstatin, leupeptin, and phosphoramidon and using high pressure such as minilab and French press, and cell-free An extract can be obtained.

From cell-free extracts, fractionation by protein solubility (precipitation with organic solvents such as acetone and dimethyl sulfoxide, salting out with ammonium sulfate, etc.), cation exchange, anion exchange, gel filtration, hydrophobic chromatography, The enzyme of the present invention can be purified by appropriately combining affinity chromatography using chelates, dyes, antibodies and the like. For example, the cell-free extract can be electrophoretically purified to almost a single band by combining column chromatography such as Blue-Sepharose, Phenyl-Sepharose, Resource Q (all manufactured by GE Healthcare). .
The molecular weight of StyA derived from ST-5 strain deduced from the amino acid sequence is 46,941 Da. The molecular weight of StyA derived from ST-10 strain deduced from the amino acid sequence is 47,290 Da. It is presumed that both StyA derived from ST-5 strain and StyA derived from ST-10 strain are monomers.

Rhodococcus sp.ST-5 strain and Rhodococcus sp.ST-10 strain isolated by the present inventors that produce the styrene monooxygenase of the present invention are patent microorganisms by the Independent Administrative Institution Product Evaluation Technology Base Organization. Deposited at the Deposit Center. The contents specifying the deposit are described below.
Rhodococcus sp.ST-5 strain (1) Depositary name: National Institute of Technology and Evaluation (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
(3) Accession number: NITE P-974
(4) Identification display: ST-5
(5) Date of deposit: August 27, 2010 Rhodococcus sp. ST-10 strain (1) Depositor name: National Institute of Technology and Evaluation (2) Contact information: Kazusa-Kama, Kisarazu City, Chiba Prefecture 292-0818 2-5-8 feet
(3) Accession number: NITE P-975
(4) Identification display: ST-10
(5) Deposit date: August 27, 2010

Polynucleotide In the present invention, the polynucleotide can be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA and RNA. The polynucleotide of the present invention can also be a DNA-RNA chimeric molecule. The polynucleotide of the present invention may be a single strand consisting only of a coding strand encoding styrene monooxygenase, or may have a double-stranded structure consisting of the coding strand and its complementary strand. .

  The polynucleotide encoding the styrene monooxygenase of the present invention can be obtained from a microorganism. As the microorganism, a microorganism belonging to the genus Rhodococcus is preferable. More specifically, Rhodococcus opacus and Rhodococcus erythropolis are preferable as microorganisms for obtaining the polynucleotide of the present invention. For example, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 was cloned from Rhodococcus opacus ST-5 strain. The base sequence shown in SEQ ID NO: 1 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. Further, for example, a polynucleotide containing the base sequence set forth in SEQ ID NO: 3 was cloned from Rhodococcus erythropolis ST-10 strain. The base sequence shown in SEQ ID NO: 3 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 4. A polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 is a preferred polynucleotide in the present invention.

  The polynucleotide of the present invention includes not only a gene cloned from Rhodococcus opacus ST-5 strain or Rhodococcus erythropolis ST-10 strain but also a homologue of the gene. Examples of such a gene homolog include a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3. In addition to the polynucleotide encoding styrene monooxygenase, a polynucleotide containing an appropriate control sequence so as not to affect the reading frame, and a polynucleotide encoding a fusion protein of styrene monooxygenase modified with an appropriate peptide sequence, It can be mentioned as a suitable example.

  Moreover, the homolog contained in the polynucleotide of the present invention includes a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4. Such a polynucleotide encodes the amino acid sequence shown in SEQ ID NO: 2 or 4 due to the degeneracy of the genetic code, in addition to the gene containing the nucleotide sequence shown in SEQ ID NO: 1 or 3. Alternatively, a polynucleotide having a base sequence different from the base sequence of 3 is included.

This time, a gene encoding styrene monooxygenase having the nucleotide sequence of SEQ ID NO: 1 or 3 was obtained. A homolog can be screened using, for example, a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 3 as a probe. Alternatively, a base sequence selected from the base sequences of SEQ ID NO: 1 or 3 can be used as a probe. As a screening method using a probe, the following known hybridization method can be used.
Colony hybridization,
Plaque hybridization,
Southern blotting screening can be performed using chromosomal DNA or cDNA libraries of Rhodococcus opacus, Rhodococcus erythropolis and other microorganisms belonging to the genus Rhodococcus, or other species.

The polynucleotide of the present invention can also be obtained by PCR using a chromosomal DNA or cDNA library of an enzyme-producing strain as a template. Primers for PCR can be designed based on the nucleotide sequence of SEQ ID NO: 1 or 3. If the DNA obtained by PCR is a fragment, its full-length sequence can be further determined based on the base sequence of the DNA fragment. For example, a full-length sequence can be determined based on fragment sequence information using inverse PCR (Genetics (1988) 120: 621-3). Inverse PCR is PCR that uses, as a template, DNA that has been partially clarified by digestion with an appropriate restriction enzyme and then cyclized by self-cyclization. By using a primer specific to a known region inside the fragment sequence, a region whose base sequence is unknown before and after that can be amplified.
Alternatively, the polynucleotide of the present invention can also be obtained by the RACE method (Rapid Amplification of cDNA End; “PCR Experiment Manual”, HBJ Publishing Bureau, p25-33).

  The polynucleotide homologue in the present invention is a polynucleotide that can hybridize with a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3 under highly stringent conditions, and has a styrene monooxygenase activity. Also included are polynucleotides that encode proteins having:

  In the present invention, highly stringent conditions are, for example, ECL direct nucleic acid labeling and detection system (manufactured by Amersham Pharmacia Biotech) using conditions described in the manual (for example, wash: 42 ° C., 0.5 × SSC). Hybridization in primary wash buffer). More specific “highly stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions. More preferably, the conditions are 65 ° C., 0.1 × SSC and 0.1% SDS. In addition to these temperatures and salt concentrations, multiple factors including probe concentration, probe length, and reaction time affect the stringency of hybridization. Those skilled in the art can adjust stringency by appropriately selecting these elements.

For example, the base sequence of the probe used for hybridization can be selected from the base sequences described in SEQ ID NO: 1 or 3. For example, a polynucleotide having at least 20, preferably at least 30, for example 40, 60 or 100 consecutive base sequences can be used as a probe. Alternatively, a polynucleotide having the full length of the base sequence described in SEQ ID NO: 1 or 3 can be used as a probe.
For details on the hybridization procedure, see `` Molecular Cloning, A Laboratory Manual 2nd ed. '' (Cold Spring Harbor Press (1989), especially Sections 9.47-9.58), `` Current Protocols in Molecular Biology '' (John Wiley & Sons (1987 -1997), especially Sections 6.3-6.4), "DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed." (Oxford University (1995), especially Section 2.10).

A polynucleotide capable of hybridizing under highly stringent conditions is likely to encode an amino acid sequence having high identity (homology) with the amino acid sequence set forth in SEQ ID NO: 2 or 4. Specifically, it is at least 70%, preferably at least 80%, more preferably 90% or more, further preferably 95% or 98%, particularly preferably 99% or more with the amino acid sequence shown in SEQ ID NO: 2 or 4. Polynucleotides encoding amino acid sequences having identity are included in preferred polynucleotides in the present invention. Such proteins with high identity are likely to have the same or similar activities.
Protein identity can be searched using databases on amino acid sequences. For example, a database in which sequence information is accumulated in amino acids of proteins such as SWISS-PROT, PIR, and DAD can be used. Alternatively, DNA identity can be searched using a database in which base sequence information is accumulated. Databases relating to DNA such as DDBJ, EMBL or GenBank are known. In these databases, amino acid sequence information predicted based on the DNA base sequence can also be used. Various kinds of sequence information can be searched by using programs such as BLAST and FASTA for these databases and the like. Any of the databases exemplified here can be used through the Internet (for example, http://www.ncbi.nlm.nih.gov.).

  Furthermore, the homologue of the polynucleotide of the present invention includes an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and styrene. A polynucleotide encoding a protein having monooxygenase activity can be mentioned. Those skilled in the art can obtain homologues of such polynucleotides by appropriately introducing substitutions, deletions, insertions, and / or addition mutations based on a certain base sequence. For example, a site-specific mutagenesis method (Nucleic Acids Res (1982) 10: 6487; Methods in Enzymol (1983) 100: 448; Molecular Cloning 2nd Edt) , Cold Spring Harbor Laboratory Press (1989); PCR A Practical Approach, IRL Press (1991) pp. 200) can be used to introduce any mutation.

When substituting amino acid residues in proteins, it is particularly preferable to perform so-called conservative amino acid substitutions with amino acids having similar side chain chemical properties. Amino acids are classified according to their side chain chemistry, for example:
(1) Neutral hydrophobic side chain (alanine, tryptophan, valine, phenylalanine, proline, methionine, leucine);
(2) Neutral polar side chain (asparagine, glycine, glutamine, cysteine, serine, tyrosine, threonine);
(3) basic side chains (arginine, histidine, lysine);
(4) acidic side chains (aspartic acid, glutamic acid);
(5) aliphatic side chain (alanine, isoleucine, glycine, valine, leucine);
(6) Aliphatic hydroxyl side chain (serine, threonine);
(7) amine-containing side chains (asparagine, arginine, glutamine, histidine, lysine);
(8) aromatic side chains (tyrosine, tryptophan, phenylalanine); and
(9) Sulfur-containing side chains (cysteine, methionine).

  In other words, mutual substitution of amino acid residues constituting each of these groups is called conservative substitution. In the present invention, the number and location of “amino acid substitutions, deletions, insertions and / or additions of one or more amino acids” are not limited as long as the protein encoded by the DNA has styrene monooxygenase activity. The number of amino acid residues that are allowed to mutate is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids. More specifically, in the amino acid sequence of SEQ ID NO: 2 or 4, variation of amino acid residues usually within 50, for example, within 20, more preferably within 5, is allowed.

  As described above, the homologue of the polynucleotide of the present invention comprises an amino acid sequence in which one or more amino acids are deleted from the amino acid sequence shown in SEQ ID NO: 2 or 4, and has a styrene monooxygenase activity. Polynucleotides encoding proteins are included. Polynucleotides containing such deletions include polynucleotides that encode “parts” of proteins consisting of the amino acid sequence of SEQ ID NO: 2 or 4.

  As long as the protein encoded by the polynucleotide retains the biological activity of the original protein (styrene monooxygenase activity), deletion, substitution, addition and / or insertion of amino acid residues in the amino acid sequence is allowed. The Here, maintaining biological activity means maintaining the ability to catalyze at least one reaction catalyzed by the original enzyme. “Maintaining activity” includes not only the same activity level, but also higher activity. Further, even when the level of activity decreases, a substantially equivalent activity is included in the maintenance of activity. Substantially equivalent refers to, for example, 50% to 100%, usually 70 to 100%, preferably 80 to 100%, more preferably 90, or 95 to 100% of the original activity. The method for evaluating the biological activity of a protein in the present invention, that is, the styrene monooxygenase activity has already been described.

  The polynucleotide of the present invention includes genomic DNA cloned from nature and cDNA, as well as polynucleotides obtained by synthesis. The polynucleotide of the present invention can be synthesized by a well-known technique based on the sequence information described in SEQ ID NO: 1 or 3, for example.

  The enzyme of the present invention can also be obtained by genetic engineering techniques or chemical synthesis methods. For example, in vitro translation (Dasso and Jackson, Nucleic Acids Res (1989) 17: 3129-44) is known as a method for producing a protein in a test tube not containing cells. In addition, the polynucleotide of the present invention can be incorporated into an appropriate expression vector, transformed into a host in which the expression vector is expressed, and a desired protein can be obtained from the host cell. A method for producing a protein using such a host-vector system will be specifically described in the section “Recombinant Vector and Transformant” below. These known protein production methods can be used as a method for obtaining the protein of the present invention.

  Proteins produced by genetic engineering techniques are recovered from biological materials containing the proteins. For example, when the protein is secreted outside the host cell, the protein is recovered from the medium in which the cell is cultured. When the host is a transgenic organism, the target protein can be recovered from the body fluid. Alternatively, when the protein is produced intracellularly, the protein is recovered from the lysate obtained by lysing the cell.

The recovered protein can be purified by the same means as when the protein is purified from cells that naturally produce the protein. In other words, known purification methods such as salting out, distillation, various chromatography, gel electrophoresis, gel filtration, ultrafiltration, recrystallization, acid extraction, dialysis, immunoprecipitation, solvent precipitation, solvent extraction, ammonium sulfate or ethanol precipitation, etc. In combination, the target protein can be purified. A person skilled in the art can use, for example, the following various types of chromatography in combination. For these chromatography, liquid phase chromatography systems such as HPLC and FPLC can be used.
Affinity chromatography,
Ion exchange chromatography such as anion or cation exchange,
Reverse phase chromatography,
Adsorption chromatography,
Gel filtration,
Hydrophobic chromatography,
Hydroxyapatite chromatography

  Further, if the protein of the present invention (styrene monooxygenase) is expressed as a fusion protein with a tag, it can be recovered using a column that binds to the tag. For example, a fusion protein having a GST tag can be easily separated using a glutathione column. Alternatively, the fusion protein with a histidine tag can be purified using a nickel column. A protease recognition sequence can be inserted between the tag and the protein (styrene monooxygenase). For example, thrombin or factor Xa can be used as the protease. After binding the fusion protein to the column, the column is washed if necessary. Subsequently, when these proteases are allowed to act, the target protein is cleaved from the tag. Thereafter, the target protein can be easily purified by recovering the cleaved protein.

Recombinant Vector and Transformant A styrene monooxygenase expression vector is provided by inserting the polynucleotide of the present invention into a known expression vector. That is, the present invention relates to a recombinant vector containing the polynucleotide of the present invention. Examples of suitable vectors include various vectors such as plasmids, cosmids, viruses, bacteriophages and the like (Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons). (1987)). Preferred vectors of the present invention include, but are not limited to, for example, E. coli BL21 (DE3) in which a gene encoding styrene monooxygenase is inserted into an expression vector pET in E. coli so that it can be expressed. It is done.

  The recombinant vector of the present invention can be constructed according to techniques commonly used in the fields of molecular biology, biotechnology and genetic engineering (Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989). Current Protocols in Molecular Biology, John Wiley & Sons (1987)). In order to express the polynucleotide of the present invention using a microorganism or the like as a host, it is first necessary to introduce the DNA into a plasmid vector or a phage vector stably present in the microorganism, and to transcribe and translate the genetic information. There is. For that purpose, a promoter is usually arranged 5 'upstream of the polynucleotide of the present invention. A promoter is a unit that controls transcription and translation.

And it is preferable to arrange | position a terminator 3 'downstream of the polynucleotide of this invention. As the promoter and terminator, those that function in the microorganism used as the host can be selected. Control sequences such as vectors, promoters and terminators that can be used in various microorganisms can be referred to in “Basic Course of Microbiology 8 Genetic Engineering” (Kyoritsu Shuppan). Particularly, yeast regulatory sequences are described in detail in Adv Biochem Eng (1990) 43: 75-102 and Yeast (1992) 8: 423-88. In addition, control sequences necessary for transcription and / or translation such as enhancer, operator sequence, initiation signal, polyadenylation signal, and ribosome binding site can be incorporated as necessary.
The vector of the present invention preferably contains all the components of the regulatory sequences required for the expression of the inserted polynucleotide of the present invention. Furthermore, the vector of the present invention can contain a selection marker for selecting a host cell into which the vector has been introduced.

  In the present invention, a sequence encoding a signal peptide can also be added to the polynucleotide of the present invention. By adding a signal peptide, proteins expressed in the host cell can be transferred to the endoplasmic reticulum lumen. Alternatively, when a gram-negative bacterium is used as a host, a protein expressed in the host cell can be transferred into the periplasm or out of the cell by a signal peptide. Any signal peptide that can function in the host cell utilized can be utilized. Therefore, a signal peptide derived from a different species for the host can be used. Furthermore, a linker, an initiation codon (ATG), a termination codon (TAA, TAG or TGA), etc. can be added when introducing the polynucleotide of the present invention into a vector, if necessary.

The host for expressing the polynucleotide of the present invention is not particularly limited as long as it is an organism that can be transformed by a recombinant vector containing the polynucleotide and can express a protein having styrene monooxygenase activity. . The present invention provides a transformant (transformed cell) transformed with the polynucleotide of the present invention or the vector of the present invention. Examples of the microorganism that is a target of the transformant of the present invention include the following microorganisms.
(1) Bacteria for which host vector systems have been developed: Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus ( Corynebacterium genus, Streptococcus genus, Lactobacillus genus, etc.
(2) Actinomycetes, Rhodococcus genus, Streptomyces genus, etc. for which host vector systems have been developed
(3) Yeast, Saccharomyces genus, Kluyveromyces genus, Schizosaccharomyces genus, Zygosaccharomyces genus, Yarrowia genus, Trichosporon (Trichosporon) genus, Rhodosporidium genus, Pichia genus, Candida genus, etc.
(4) Host vectors have been developed, such as molds, Neurospora, Aspergillus, Cephalosporium, Trichoderma

  For example, in the genus Escherichia, particularly Escherichia coli, plasmid vectors such as pET, pBR, and pUC can be used, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp fusion) ), Promoters derived from λ phage PL, PR, etc. can be used. Moreover, as the terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used. In particular, a vector pSE420D (described in JP-A-2000-189170) obtained by partially modifying a multicloning site of commercially available pSE420 (manufactured by Invitrogen) is a suitable vector when an Escherichia bacterium is used as a host.

  In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors. When these vectors are used, the polynucleotide of the present invention can be integrated into the host chromosome. As the promoter and terminator, apr (alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used.

  In the genus Pseudomonas, host vector systems have been developed for Pseudomonas putida, Pseudomonas cepacia, and the like. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240 based on the plasmid TOL plasmid, which is involved in the degradation of toluene compounds, can be used, and lipase (special Kaihei 5-284973) Gene-derived ones can be used.

  In the genus Brevibacterium, particularly Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene (1985) 39: 281) can be used. As the promoter / terminator, the promoter and terminator used in Escherichia coli can be used as they are.

  In the genus Corynebacterium, in particular, Corynebacterium glutamicum, plasmid vectors such as pCS11 (Japanese Patent Laid-Open No. 57-183799) and pCB101 (Mol Gen Genet (1984) 196: 175) can be used.

  In the genus Streptococcus, pHV1301 (FEMS Microbiol Lett (1985) 26: 239), pGK1 (Appl Environ Microbiol (1985) 50:94) and the like can be used as plasmid vectors.

  In the genus Lactobacillus, pAMβ1 (J Bacteriol (1979) 137: 614) developed for Streptococcus can be used as a vector, and a promoter used in Escherichia coli can be used.

  In the genus Rhodococcus, plasmid vectors isolated from Rhodococcus rhodochrous can be used in addition to Rhodococcus opacus and Rhodococcus erythropolis (J Gen Microbiol (1992) 138: 1003).

  In the genus Streptomyces, a plasmid can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, pIJ486 (Mol Gen Genet (1986) 203: 468-78), pKC1064 (Gene (1991) 103: 97-9), pUWL-KS (Gene (1995) 165: 149-50) can be used. A similar plasmid can also be used in Streptomyces virginiae (Actinomycetol (1997) 11: 46-53).

  In the genus Saccharomyces, particularly Saccharomyces cerevisiae, YRp, YEp, YCp, and YIp plasmids can be used. In addition, an integration vector (such as EP537456) using homologous recombination with ribosomal DNA present in multiple copies in a chromosome is extremely useful because it can introduce a gene in multiple copies and can stably hold the gene. ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) Promoter terminators such as are available.

  In the genus Klyberomyces, particularly Kluyveromyces lactis, a 2 μm-derived plasmid derived from Saccharomyces cerevisiae, a pKD1-based plasmid (J Bacteriol (1981) 145: 382-90), pGKl1 involved in killer activity A plasmid derived from the gene, an autonomously proliferating gene KARS plasmid in the genus Kleiberymyces, a vector plasmid (EP537456, etc.) that can be integrated into the chromosome by homologous recombination with ribosomal DNA and the like can be used. In addition, promoter / terminators derived from ADH, PGK and the like can be used.

  In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selection markers that complement the auxotrophy derived from Saccharomyces cerevisiae are available. (Mol Cell Biol (1986) 6:80). Further, an ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J (1987) 6: 729). In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

  In the genus Tigosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13: 4267 (1985)) derived from Zygosaccharomyces rouxii can be used, and the PHO5 promoter derived from Saccharomyces cerevisiae and Tigo A promoter of Saccharomyces rouxi-derived GAP-Zr (glyceraldehyde-3-phosphate dehydrogenase) (Agri Biol Chem (1990) 54: 2521) and the like can be used.

  In the genus Pichia, a host vector system using Pichia angusta (former name: Hansenula polymorpha) has been developed. As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but they are relatively unstable, so multicopy integration into the chromosome is effective (Yeast (1991) 7 : 431-43). In addition, AOX (alcohol oxidase), FDH (formate dehydrogenase) promoter, etc. induced by methanol or the like can be used. For Pichia pastoris, etc., host vector systems using genes (PARS1, PARS2), etc. involved in Pichia-derived autonomous replication have been developed (Mol Cell Biol (1985) 5: 3376). A strong promoter such as AOX that can be induced by concentration culture and methanol can be used (Nucleic Acids Res (1987) 15: 3859).

  In the genus Candida, host vector systems have been developed for Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis, etc. ing. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri Biol Chem (1987) 51: 1587), and a vector using this has been developed. Further, in Candida utilis, a strong promoter has been developed for a chromosome integration type vector (Japanese Patent Laid-Open No. 08-173170).

  In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae and the like have been most studied. Plasmids using these as hosts can be used, and integration of desired genes into chromosomes can be performed. Promoters derived from extracellular proteases and amylases are also available (Trends in Biotechnology (1989) 7: 283-7).

  In the genus Trichoderma, a host vector system using Trichoderma reesei has been developed, and an extracellular cellulase gene-derived promoter or the like can be used (Biotechnology (1989) 7: 596-603).

  In addition to microorganisms, various host vector systems using plants and animals as hosts have been developed. For example, insect vector systems using moths (Nature (1985) 315: 592-4) and plant vector systems such as rapeseed, corn and potato have been developed as systems for expressing large amounts of heterologous proteins. Available to:

  Introduction of the polynucleotide of the present invention into a vector can be performed by a ligase reaction using a restriction enzyme site (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 11.4-11.11; Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989) Section 5.61-5.63). In addition, considering the codon usage frequency of the host to be used, the polynucleotide sequence of the present invention may be modified as necessary to design a vector with high expression efficiency (Grantham et al., Nucleic Acids Res ( 1981) 9: r43-74).

  As described above, various cells have been established as host cell lines. Methods for introducing an expression vector suitable for each cell line are also known, and those skilled in the art can select a method suitable for each selected host cell. For example, for prokaryotic cells, calcium treatment, transformation by electroporation and the like are known. For plant cells, methods using Agrobacterium are known, and for mammalian cells, calcium phosphate precipitation can be exemplified. The present invention is not particularly limited to these methods, and other known nuclear microinjection, protoplast fusion, DEAE-dextran method, cell fusion, electric pulse perforation method, lipofectamine method (GIBCO BRL) depending on the selected host. The expression vector can be introduced by various known methods including a method using FuGENE6 reagent (Boehringer-Mannheim).

  By culturing the transformant transformed with the recombinant vector containing the polynucleotide of the present invention as described above, the protein having the styrene monooxygenase activity of the present invention can be produced. Therefore, as a preferred embodiment of the present invention, there is provided a method for producing an optically active styrene monooxygenase, comprising the steps of culturing the transformant of the present invention and recovering the protein encoded by the polynucleotide from the culture. Provided. The method for culturing the transformant is not particularly limited, and it is desirable to select conditions such as a medium, temperature, and time that are suitable for the growth of each selected host cell and most suitable for the production of the enzyme of the present invention. For example, the conditions described in the examples can be mentioned, but the present invention is not limited thereto.

TECHNICAL FIELD The present invention relates to a method for producing an optically active styrene oxide and / or an optically active styrene analog oxide. The method is characterized in that the vinyl group of styrene and / or its analogue is oxidized to an oxiranyl group by styrene monooxygenase. The method for producing an oxide of styrene oxide and / or styrene analog of the present invention comprises a step of allowing styrene monooxygenase to act on styrene and / or an analog thereof, and a step of recovering the produced oxide.

Styrene monooxygenase and the substrate are allowed to act under conditions that allow oxidation of the substrate. Specifically, in the oxidation reaction of the present invention, it is necessary to add FADH ₂ (or reduced FAD) as a coenzyme. The oxidation reaction of the present invention is shown below. In the oxidation reaction of the present invention, coenzyme FADH ₂ is oxidized to produce FAD, and at the same time, water is produced.

The styrene monooxygenase can be used in the form of an enzyme molecule, a processed product thereof, a culture containing the enzyme molecule, a microorganism such as a transformant producing the enzyme, or a processed product thereof.
The processed product of the microorganism of the transformant that produces styrene monooxygenase specifically includes a microorganism whose cell membrane permeability has been changed by treatment with an organic solvent such as a surfactant or toluene, or a glass bead or enzyme treatment. A cell-free extract obtained by disrupting the body, a partially purified product, a highly purified product, a product immobilized on various immobilization carriers, and the like are included.

  By reacting such styrene monooxygenase with styrene and / or an analog thereof in the reaction solution, the target enzyme reaction can be carried out to obtain an oxide of styrene oxide and / or styrene analog. The mode of action of the enzyme and the reaction solution is not limited to these specific examples. In the present invention, “action” can also be expressed as “contact”.

The reaction solution is preferably a solution obtained by dissolving FADH ₂ , which is a coenzyme necessary for a substrate and an enzyme reaction, in an appropriate solvent that provides a desirable environment for the expression of enzyme activity. As such a solvent, for example, an organic solvent which is difficult to dissolve in water or water, for example, an organic solvent such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary butyl ester, or , A two-phase system with an aqueous medium, or a mixed system with an organic solvent dissolved in water, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide and the like.

Examples of the styrene analog in the present invention include compounds in which at least one of the 2-position, 3-position and 4-position of styrene is substituted with a halogen. Halogen includes, but is not limited to, fluorine, chlorine, bromine and the like. The following compounds can be illustrated as such a styrene analog.
2-fluorostyrene, 3-fluorostyrene, 4-fluorostyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2-bromostyrene, 3-bromostyrene, 4-bromostyrene or in the present invention Other examples of styrene analogs may include the following compounds:
・ Indene ・ p-aminostyrene ・ p-methoxystyrene ・ trans-β-methylstyrene ・ 4-vinyl-1-chlorohexene ・ N, N-dimethyl-1-naphthylamine ・ cinnamaldehyde Oxidation by contacting with a styrene monooxygenase can yield an oxide of a styrene analog. Specifically, for example, 2-fluorostyrene oxide can be obtained by oxidizing 2-fluorostyrene with the styrene monooxygenase of the present invention. Alternatively, for example, indene oxide can be obtained by oxidizing indene with the styrene monooxygenase of the present invention.

The FAD produced from FADH ₂ in association with the oxidation reaction, regeneration of the FADH ₂ is (such as respiratory activity) FAD reducing ability possessed by the microorganism can be carried out using. Also it is carried out by adding microorganisms and processed product thereof having an ability to produce FADH ₂ from FAD, the enzyme reaction system. For example, FADH ₂ can be regenerated using a microorganism containing flavin oxidoreductase, a processed product thereof, and a partially purified or purified enzyme. The components constituting the reaction necessary for the regeneration of FADH ₂ are added to the reaction system for the production of styrene oxide or styrene analog oxide according to the present invention, added in an immobilized state, or exchange of FAD +. Can be contacted through a possible membrane.
Alternatively, a microorganism transfected with DNA encoding flavin oxidoreductase or a processed product thereof can be added to the reaction system for the oxidation reaction of the present invention.

  The reaction of the present invention can also be performed using an immobilized enzyme, a membrane reactor, or the like.

  In the method for producing an oxide of styrene oxide or a styrene analog, the reaction temperature is preferably 4 to 60 ° C., more preferably 15 to 37 ° C., the pH is preferably 4 to 11, more preferably pH 5 to 9, and the substrate concentration. Is preferably 0.01 to 90%, more preferably 0.1 to 30%.

Coenzyme FADH ₂ can be added to the reaction system as necessary, preferably 0.001 mM to 100 mM, more preferably 0.01 to 10 mM.
The substrate can be added all at once at the start of the reaction, but it is desirable to add it continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high. The substrate concentration can be, for example, 0.01 g / L to 500 g / L, preferably 0.1 g / L to 100 g / L, more preferably 0.5 g / L to 10 g / L, but is not limited thereto.

An enzyme for regenerating FADH ₂ , such as flavin oxidoreductase, is preferably added in an amount of 0.1 to 100 times, more preferably about 0.5 to 20 times in terms of enzyme activity compared to the oxidase of the present invention. it can.

  Recovery of oxides of styrene oxide or styrene analog produced by the oxidation reaction of the present invention is performed by removing cells and proteins by centrifugation or membrane filtration, separating proteins by membrane treatment, etc., ethyl acetate, butyl acetate, chloroform, methyl -T-butyl ether, n-butanol, sec-butanol, t-butanol, isobutanol, n-pentanol, sec-pentanol, t-pentanol, isopentanol, etc., solvent extraction, distillation, ethyl acetate, butyl acetate Crystallization by xylene, toluene, mesitylene, chlorobenzene, chloroform, n-hexane, isooctane, diisopropyl ether, methyl-t-butyl ether, and the like. Or it can also collect | recover as hydrochloride produced | generated by dripping hydrochloric acid to an extraction solvent liquid. The desired product can be further purified by silica gel column chromatography and distillation if necessary.

Method for Decomposing Styrene or an Analogue Further The present invention relates to a method for decomposing styrene or an analog thereof, comprising the step of contacting the styrene monooxygenase of the present invention with styrene or an analog thereof. As described above, the styrene monooxygenase of the present invention oxidizes the vinyl group of styrene or a derivative thereof to an oxiranyl group. Accordingly, the styrene monooxygenase of the present invention can degrade styrene or its analogs.
In the present invention, decomposition means that the original compound is converted into a different compound. Examples of the conversion include oxidation. Therefore, in the present invention, the decomposition method can also be expressed as an oxidation method. Alternatively, it can be expressed as a method of reducing the amount or concentration of styrene or an analog thereof.
Examples of styrene analogs include those described above. The form of contact between the styrene monooxygenase and the substrate has already been described.
It should be noted that all documents cited in this specification are incorporated herein by reference.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
Materials and methods
(1) Bacterial strains and vectors Styrene anabolic bacteria ST-5 and ST-10 strains were used as the origin of chromosomal DNA for isolation of styrene metabolic genes. These microorganisms were previously identified as Corynebacterium sp. ST-5 and ST-10 strains (Itoh, N., K. Yoshida, and K. Okada. 1996. Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism. Biosci. Biotech. Biochem. 60: 1826-1830.). Recently, Makino et al. Re-identified the ST-10 strain as Rhodococcus sp. ST-10 (Makino, Y., K. Inoue, T. Dairi, and N. Itoh. 2005. Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol. Appl. Environ. Microbiol. 71: 4713-4720). The present inventors also re-identified the ST-5 strain by the method described by Makino et al. The results showed that the ST-5 strain has typical characteristics of Rhodococcus. Therefore, the inventors concluded that this microorganism should be identified as Rhodococcus sp. ST-5 strain. E. coli strain JM109 and plasmid vector pGEM-T Easy (Plomega) were used in the DNA manipulation.

(2) Culture conditions Rhodococcus sp. ST-5 and ST-10 were shaken at 30 ° C. for 2 days in PY medium (0.5% yeast extract, 0.5% bactopeptone, pH 7.0). Cultured. 10 μl of culture broth was added to agar plates (0.3 (w / v)% (NH ₄ ) ₂ SO ₄ , 0.3% KH ₂ PO ₄ , 0.1% NaCl, 0.02% MgSO ₄ 7H ₂ O, and 1.5% agar, pH 7. 0) and cultured in a styrene atmosphere at 30 ° C. for 4 days (Itoh, N., K. Yoshida, and K. Okada. 1996. Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism. Biosci. Biotech. Biochem. 60: 1826-1830).

(3) Cloning of styrene metabolism gene
Standard techniques were used for DNA manipulation (Sambrook, J., and WD Russell. 2001. “Molecular cloning, a laboratory manual” 3rd ed. Cold Spring Harbor Laboratory Press.). The collected cells of ST-5 and ST-10 were treated with lysozyme, followed by Eulberg et al. (Eulberg, D., LA Golovleva, and M. Schlomann. 1997. Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP. J Bacteriol. 179: 370-381) was used to prepare genomic DNA. The prepared genomic DNA was used for PCR template and Southern blot analysis.
Partial fragments encoding styA gene and styC gene were amplified by degenerate PCR. The primers used for the amplification of styA and styC are shown in Table 1. These oligonucleotide primers were designed based on partial amino acid sequences conserved in styrene monooxygenase and styrene oxide isomerase, respectively. In order to obtain the full length of a gene involved in styrene metabolism, TAIL (thermal asymmetric interlaced) PCR was performed using the partial nucleotide sequence of the styA gene or styC gene that was revealed. TAIL-PCR was performed as described by Liu et al. (Liu, YG, and RF Whittier. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674- 681). The adapter primers used for TAIL-PCR and known sequence specific primers are shown in Table 1. Primary and secondary PCR products were diluted and used as templates for secondary and tertiary PCR, respectively. Amplified fragments were purified by separation and extraction by agarose gel electrophoresis. The purified fragment was cloned into pGEM-T Easy and the nucleotide sequence was determined using a capillary DNA sequencer 310 (Applied Biosystems, Tokyo, Japan).

(4) Southern blot analysis
Rhodococcus sp. ST-10 genomic DNA was digested with several restriction enzymes and separated by agarose gel electrophoresis. The isolated DNA was transferred to Hybond N ⁺ nylon membrane (GE Healthcare UK Ltd, Buckinghamshire, UK) and baked at 80 ° C. for 2 hours. Styrene monooxygenase gene fragment (1.2 kbp) derived from ST-10 strain and styrene oxide isomerase gene fragment (0.6 kbp) derived from ST-5 strain were amplified by PCR and used as probes for hybridization. Each fragment was labeled with α- ³² P-dCTP using Random Primer DNA Labeling Kit Ver. 2 (TaKaRa, Shiga, Japan). The labeling operation was performed according to the product protocol. Prehybridization and hybridization were performed in a hybridization buffer consisting of 5 × SSC, 0.1% SDS, 5 × Denharz solution, 40% formamide. 1 ng of labeled probe DNA was added to the membrane and incubated overnight at 45 ° C. The membrane was washed twice with 2 × SSC buffer for 10 minutes at 45 ° C. and then twice with 2 × SSC containing 0.1% SDS for 10 minutes at 45 ° C. Detection of labeled DNA hybridized to the membrane was performed using a BAS-3000 image analyzer (Fujifilm, Tokyo, Japan).

(5) Extraction of metabolites by stationary cell reaction Cell bodies cultured in a styrene atmosphere are suspended in 2 ml of 50 mM potassium phosphate buffer (pH 7.0) and centrifuged (8000 × g, 20 minutes). Collected. The collected cells were resuspended in the same buffer. Next, a static cell reaction was carried out using a reaction mixture consisting of 4 ml of a cell suspension (OD ₆₀₀ = 1) and 50 mg of each substrate. The reaction mixture was added to a 22 ml vial and sealed with a silicone septum and incubated at 30 ° C. for 20 hours at 170 rpm. After the incubation, 2 ml of ethyl acetate was added to and mixed with the reaction mixture, and the reaction product was extracted. The extract was analyzed by gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC).

(6) Analysis conditions of GC-MS and HPLC GC-MS analysis of the product by stationary cell reaction was performed using GC-17A gas chromatography system and Shimadzu QP-5000 mass spectrometer. A sample of 1 μl extracted with ethyl acetate was injected into an HP-1MS capillary column (J & W Scientific Inc .; 30 m × inner diameter 0.25 mm, film width 0.25 μm). The carrier gas (He) flow rate was 1.7 ml / min (100 kPa), the split ratio was 26, and the linear velocity was 47.4 cm / s. The temperature program for the column oven was: 50 ° C, 5 minutes; ramped to 220 ° C at 10 ° C / min; 220 ° C, 5 minutes. Mass spectra were acquired at 1.5 kV using an electron impact ion source (EI, 200 ° C.). Each reaction intermediate was identified by comparison with the retention time and mass-to-charge ratio (m / z) of the standard compound.
The HPLC analysis of the product by the stationary cell reaction was carried out using 10 mM ammonium acetate as mobile phase A, acetonitrile as mobile phase B, a flow rate of 0.5 ml / min, and a column temperature of 40 ° C. After injecting the sample (10 μl), mobile phase B was maintained at 10% for 8 minutes, then increased to 90% over 20 minutes and maintained for 10 minutes. The product was monitored at 254 nm.
Identification of the reaction product of phenylacetyl-CoA ligase was performed using a Shimadzu Prominence GE HPLC system. For analysis, a Cadenza CD-C18 column (Imtakt, Kyoto, Japan) was used. The mobile phase used was 0.2M KPB (pH 4.5) containing 10% (v / v) isopropyl alcohol. The mobile phase flow rate was 0.5 ml / min and the column was maintained at 40 ° C. The product was monitored at 254 nm.

(7) Preparation of cell-free crude extract and enzyme assay Cell-free crude extract was obtained from cells cultured using gaseous styrene as a single carbon source. The collected cells were suspended in 50 mM potassium phosphate buffer (pH 7.0) and washed twice. The cells were resuspended in 50 mM potassium phosphate buffer (pH 7.0) containing 10% glycerol. The cell suspension was subjected to ultrasonic treatment for 1 minute each 5 times and centrifuged (16,000 × g, 10 minutes, 4 ° C.). The supernatant was dialyzed against the same buffer and used for enzyme assay. Phenylacetyl-CoA ligase (PCL) activity was determined by a previously reported method (Martinez-Blanco, H., A. Reglero, LB Rodriguez-Aparicio, and JM Luengo. 1990. Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. J. Biol. Chem. 265: 7084-7090).

(8) The chemical substances styrene and styrene oxide were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Phenylacetaldehyde was supplied by Toyotama International Inc. (Tokyo, Japan). All other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

[Example 1] In order to isolate and analyze the gene structural metabolic genes of styrene metabolic genes of Rhodococcus sp. ST-5 and ST-10 , the present inventors made use of styrene monooxygenase and styrene registered on the database. A highly conserved region was selected from the amino acid sequence of oxidoisomerase, and oligonucleotide primers for degenerate PCR were designed. Degenerate PCR was performed using ST-5 genomic DNA as a template, and a 260 bp fragment encoding part of styC was obtained. However, the fragment corresponding to styA was not amplified. On the other hand, when degenerate PCR was performed using the genomic DNA of ST-10 strain as a template, a partial fragment encoding styA was amplified, but the gene encoding styC was not amplified. The amino acid sequences deduced from these fragments showed significant similarity to Pseudomonas styrenolytic enzymes.
In order to obtain the full length of the styrene metabolism gene, the present inventors performed TAIL-PCR on the genomic DNA of the ST-5 and ST-10 strains. The primers used for TAIL-PCR are shown in Table 1. As a result, the present inventors were able to determine the nucleotide sequences of the approximately 4.7 kbp region of the chromosome of ST-5 strain and the 4.2 kbp region of ST-10 strain, respectively. There were 4 and 3 open reading frames (ORF) in the determined chromosomal regions of the ST-5 and ST-10 strains, respectively (FIGS. 1 and 2). Since the deduced amino acid sequence was similar to Pseudomonas styABCD, these genes were presumed to be styrene metabolism genes. The ST-5 strain had four consecutive genes corresponding to SMO, flavin oxidoreductase, SOI, and phenylacetaldehyde dehydrogenase (PAD). On the other hand, the ST-10 strain had two genes corresponding to SMO and flavin oxidoreductase. Orf3, which was present downstream of styB, showed a high similarity to a protein of unknown function in which homologs were found only in the genus Rhodococcus, such as Rhodococcus jostii RHA1. Furthermore, stem loop structures predicted to act as transcription terminators existed downstream of ST-5 strain styC and ST-10 strain styB, respectively (FIG. 1).

[Example 2] Southern blot analysis of ST-10 genomic DNA
In order to confirm the presence of the SOI gene, the present inventors performed Southern blot analysis on the chromosome of the ST-10 strain. ST-10 strain probes each 1.2kbp and 0.8kbp corresponding to styC gene styA gene and ST-5 strain, ^alpha-32 labeled with P-dCTP, ST-10 strain digested with various restriction enzymes Hybridized to genomic DNA. As a result, a significant fragment that hybridizes to the probe corresponding to the styA gene could be found, but no detectable signal was found when the styC gene was used as a probe.

[Example 3] Structural characteristics of styrene metabolism genes of ST-5 and ST-10 strains
The styA gene derived from ST-5 and ST-10 strains encoded 427 and 428 amino acid residues, and the deduced amino acid sequence showed significant similarity to the sequence of SMO from Pseudomonas sp. Y2 (60.6 % And 60.4%) (Table 2).

The deduced amino acid sequence of StyA derived from ST-5 and ST-10 strains is conserved in SMO, which is known to be involved in binding to flavin and nicotinamide adenine dinucleotide (NAD ⁺ / NADH) Contained several sequences. However, StyA of the ST-5 and ST-10 strains is a styA2B of Rhodococcus opacus 1CP (Tischler, D., D. Eulberg, S. Lankner, reported as a fusion protein of SMO and flavin oxidoreductase. SR Kaschabek, WJH van Berkel, and M. Schlomann. 2009. Identification of a novel self-sufficient styrene monooxygenase from Rhodococcus opacus 1CP. J. Bacteriol. 191: 4996-5009) (29.2% and 28.0%). Rhodococcus opacus 1CP was reported not to have a styrene metabolism gene corresponding to styC or styD in the vicinity of styA2B. These facts indicate that the previously reported Rhodococcus styrene metabolism gene is different from the gene of the present invention.

  The styB genes of ST-5 and ST-10 encoded 179 and 183 amino acid residues, respectively, and the estimated molecular weights calculated from the amino acid sequences were 19,284 Da and 19,543 Da, respectively. The deduced amino acid sequences of StyB from ST-5 and ST-10 strains contain several motifs that are highly conserved in flavin oxidoreductases and various monooxygenases. From the results of the homology search, styB derived from ST-5 and ST-10 strains were Pseudomonas sp. Y2 StyB (53.8% and 50.6%), Bacillus sp. JF8 MocB (36.5% and 36.8%) Of NAD (P) H: flavin oxidoreductase, including Geobacillus thermoglucosidasius A7 PheA2 (33.6% and 35.1%), and Rhodococcus opacus 1CP StyA2B (27.6% and 33.3%, part B) It became clear that each showed homology with several proteins belonging to the family (Table 2). This result showed that styB of ST-5 and ST-10 strains is a member of NAD (P) H: flavin oxidoreductase and constitutes a monooxygenase system consisting of two components together with StyA. .

  The styC and styD genes were observed only in the ST-5 strain. The styC gene encodes 168 amino acid residues, and the deduced amino acid sequence is significantly similar to Pseudomonas sp. Y2 SOI (69.5%), and p-cymene methylhydroxylase of Pseudomonas aureofaciens Showed moderate similarity (35.0%) (Table 2). In the open reading frame encoding styD, there were two putative start codons at bases 3068 and 3086 (FIG. 2A). From this, it was suggested that the polypeptide encoded by styD is 502 amino acids to 496 amino acids. Rhodococcus josti RHA1 ALDH (78.6%), Burkholderia sp. 383 BADH (54.4%), Azoarcus sp. BH72 ThmS2 (54.5%), Pseudomonas sp. Y2 StyD (49.9%) ), And high similarity to various aldehyde dehydrogenases such as E. coli W3110 PadA (40.1%) (Table 2). StyD has several cofactor binding motifs conserved in various aldehyde dehydrogenases (Hempel, J., H. Nicholas, and R. Lindahl. 1993. Aldehyde dehydrogenases: Widespread structural and functional diversity within a shared framework Protein Sci. 2: 1890-1900, Liu, ZJ, YJ Sun, J. Rose, YJ Chung, CD Hsial, WR Chang, I. Kuo, J. Perozich, R. Lindahl, J. Hempel, and BC Wang. 1997. The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold. Nat. Struct. Biol. 4: 317-326).

[Example 4] Analysis of intermediates of styrene metabolism by Rhodococcus sp.
ST-5 and ST-10 strains were used for static cell reaction, and the resulting intermediate was identified by GC-MS. When styrene was used as a substrate, styrene oxide was detected as a reaction product of the ST-10 strain, but phenylacetaldehyde was not detected. In contrast, the ST-5 strain accumulated only phenylacetaldehyde as a reaction product. These results were suggested to be due to differences in gene expression between the ST-5 and ST-10 strains. In ST-5 strain, styrene is converted to phenylacetaldehyde by the action of StyA, StyB, and StyC, but in ST-10 strain, styrene oxide cannot be converted to phenylacetaldehyde due to lack of StyC. It is presumed that it was accumulated in the reaction product.

  In order to elucidate the styrene utilization mechanism of the ST-10 strain, the present inventors investigated in detail the products of the ST-10 strain by the stationary cell reaction by HPLC. Various aromatic compounds including styrene oxide, acetophenone, styrene glycol, and phenylacetic acid were found in the reaction products based on styrene (FIG. 3A). As a control, styrene glycol, acetophenone, and phenylacetaldehyde were detected when styrene oxide was reacted in a buffer solution not containing ST-10 strain cells (FIG. 3B). Styrene oxide is an unstable compound and has been reported to be easily converted to other compounds in the presence of water, peroxide, or air (Liu, Z., J. Michel, Z Wang, B. Witholt, and L. Zhi. 2006. Enantioselective hydrolysis of styrene oxide with the epoxide hydrolase of Sphingomonas sp.HXN-200. Tetrahedron Asymmetry 17: 47-52, Pfaffli, P., H. Vainio, and A Hesso. 1979. Styrene and styrene oxide concentration in the air during the lamination process in the reinforced plastics industry. Scand. J. Work Environ. Health 5: 158-161). Therefore, it is speculated that acetophenone and phenylacetaldehyde were produced from styrene oxide by a spontaneous reaction. Furthermore, when incubated with the cells of ST-10 strain, phenylacetaldehyde disappeared and phenylacetic acid increased instead (FIG. 3C).

[Example 5] Identification of enzyme activity involved in the metabolism of phenylacetic acid In order to identify the degradation pathway of phenylacetic acid, the phenylacetyl-CoA ligase (PCL) activity of ST-5 and ST-10 strains was measured. . Cell-free crude extracts of ST-5 and ST-10 strains were added to a buffer containing phenylacetic acid, CoA, and ATP as substrates, and the reaction products were analyzed by HPLC. In any strain, a peak corresponding to phenylacetyl-CoA was detected at a position of 8.1 minutes in the presence of a cell-free crude extract (FIG. 4). These results indicate that all strains have PCL activity and can degrade phenylacetic acid via a pathway similar to the phenylacetic acid (PAA) degradation pathway reported for Pseudomonas bacteria.

Hereinafter, the results obtained in Examples 1 to 5 will be considered.
Rhodococcus sp. ST-5 and ST-10 (formerly Corynebacterium sp.) Can assimilate gaseous styrene and styrene oxide as a single carbon source, and these strains share the same styrene metabolic pathway as Pseudomonas It has been suggested to assimilate styrene and styrene oxide (Itoh, N., K. Yoshida, and K. Okada. 1996. Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism Biosci. Biotech. Biochem. 60: 1826-1830).

  In order to elucidate the degradation pathway of styrene in the ST-5 and ST-10 strains, the present inventors isolated a group of genes involved in styrene degradation using PCR technology.

  As shown in FIGS. 1 and 2, the present inventors determined the nucleotide sequence of the approximately 4.7 kbp chromosomal region of the ST-5 strain and the approximately 4.2 kbp region of the ST-10 strain. The sequence determined in the ST-5 strain contained 4 ORFs, and the deduced amino acid sequence was homologous to the styrenic gene styABCD reported in Pseudomonas bacteria. On the other hand, the sequence determined in the ST-10 strain has three ORFs, styAB and a protein of unknown function. Styrene metabolism genes isolated from various microorganisms including Pseudomonas bacteria are composed of four genes including SMO, flavin oxidoreductase, SOI, and phenylacetaldehyde dehydrogenase. In addition, some microorganisms have genes encoding transcriptional regulators (styRS) and substrate transporter (styE) upstream or downstream of these genes (Mooney, A., ND O'Leary, and ADW Dobson. 2006). Cloning and functional characterization of the styE gene, involved in styrene transport in Pseudomonas putida CA-3.Appl.Environ.Microbiol. 72: 1302-1309, Panke, S., B. Witholt, A. Schmid, and MG Wubbolts. 1998. Towards a biocatalyst for (S) -styrene oxide productio: characterization of the styrene degradation pathway of Pseudomonas sp. Strain VLB120. Appl. Environ. Microbiol. 64: 2032-2043, Velasco, A., S. Alonso, JL Garcia , J. Perera, and E. Diaz. 1998. Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. Strain Y2. J. Bacteriol. 180: 1063-1071). Both the ST-5 and ST-10 strains did not have a regulatory gene upstream of styA, and the ST-10 strain was found to lack styCD (FIG. 1). However, when these strains were cultured in gaseous styrene or styrene oxide, the activity of styrene monooxygenase increased, so that there may be regulatory genes that control the expression of these genes as in Pseudomonas bacteria. It was suggested. However, such a gene is not present in the region adjacent to the styrene metabolism gene.

  In order to confirm that the styrene oxide isomerase gene is not present in the chromosome of the ST-10 strain, the present inventors used a styC gene derived from the ST-5 strain as a probe to perform a Southern blot on ST-10 strain genomic DNA. Analysis was performed. As a result, a specific signal corresponding to the styC gene could not be found. On the other hand, when the styA gene was used as a probe as a control, a specific band was detected. Itoh et al. Report that the ST-10 strain does not have SOI activity but has SMO activity and PAD activity (Itoh, N., K. Yoshida, and K. Okada. 1996. Isolation and identification of styrene-degrading Corynebacterium strains, and their styrene metabolism. Biosci. Biotech. Biochem. 60: 1826-1830). These facts suggest that the ST-10 strain has styrene-degrading enzymes other than SOI and assimilate styrene oxide via an unknown pathway.

In order to elucidate the styrene utilization pathway of ST-10 strain, stationary cell reaction was performed by ST-5 strain and ST-10 strain, and the produced intermediate was identified. GC-MS analysis revealed that styrene oxide was accumulated as a reaction product of ST-10 strain, and phenylacetaldehyde was accumulated in ST-5 strain. These phenomena are consistent with the absence of a gene encoding SOI in the ST-10 strain. In previous reports, it was found that the ST-5 strain showed higher SOI activity than PAD activity (33 U / g cell and 1.5 U / g cell in wet cell weight, respectively). In the ST-5 strain, a sequence presumed to be a transcription terminator is found in the region between styC and styD (Fig. 2). Therefore, it is estimated that the expression level of styD is lower than that of other styrene metabolism genes. It is suggested that phenylacetaldehyde is accumulated as a metabolic intermediate of ST-5 strain due to low PAD activity in styrene metabolic flux. In contrast, the ST-10 strain lacks StyC, so styrene oxide accumulates as a metabolic intermediate. HPLC analysis proved that styrene oxide spontaneously isomerized to phenylacetaldehyde and acetophenone, and that ST-10 strain assimilated spontaneously isomerized phenylacetaldehyde (Figure 3). . Itoh et al. Report that styrene, styrene oxide, and 2-phenylethanol are metabolized by the ST-10 strain, but acetophenone and styrene glycol are not utilized as carbon sources. The results of the HPLC analysis are in good agreement with these facts. When styrene or styrene oxide was used as a substrate, 2-phenylethanol was observed in the reaction products of both strains. As shown in FIG. 5, phenylacetaldehyde is converted to 2-phenylethanol by the activity of phenylacetaldehyde reductase (PAR). It is suggested that 2-phenylethanol plays a role of a storage compound in the metabolic pathway of styrene. It is assumed that the 2-phenylethanol / phenylacetaldehyde ratio is controlled by the concentration of each compound and the NAD ⁺ / NADH ratio.

The phenylacetic acid metabolic pathway has been well analyzed in gram-negative bacteria such as Pseudomonas putida and E. coli (Luengo, JM, JL Garcia, and ER Olivera. 2001. The phenylacetyl-CoA catabolon: a complex catabolic unit with broad biotechnological applications. Mol. Microbiol. 39: 1434-1442). However, there is little information about the same pathway of Gram-positive bacteria. In recent years, Llorens has reported that Rhodococcus sp.RHA1 has a paa gene cluster corresponding to the phenylacetic acid metabolic pathway of Gram-negative bacteria (Navarro-Llorens, JM, MA Patrauchan, GR Stewart, JE Davies, LD Eltis , and WW Mohn. 2005. Phenylacetic acid catabolism in Rhodococcus sp. strain RHA1: a central pathway for degradation of aromatic compounds. J. Bacteriol. 187: 4497-4504). Furthermore, the analysis of the whole genome sequence of several Rhodococcus bacteria revealed that the paa gene cluster exists in the genome. The ST-5 and ST-10 strains also have PCL activity like other Rhodococcus bacteria (FIG. 4). This fact suggests that the ST-5 and ST-10 strains have the paa gene group and assimilate phenylacetic acid using the same pathway as other gram-negative bacteria.
The estimated styrene metabolism pathway in ST-5 and ST-10 strains is shown in FIG. Styrene is first oxidized to styrene oxide by SMO and subsequently converted to phenylacetaldehyde by SOI. Since the ST-10 strain lacks SOI, this reaction is presumed to have occurred by spontaneous chemical conversion. Phenylacetaldehyde is then oxidized to phenylacetic acid by PAD activity and degraded by the PAA degradation pathway. The present inventors have confirmed that styrene-degrading genes are present in the genomes of ST-5 and ST-10 strains. The ST-10 strain has an incomplete styrene metabolism pathway lacking SOI, but the ST-10 strain can assimilate styrene and styrene oxide.

[Example 6] Substrate specificity of styA derived from Rhodococcus sp. ST-5 and ST-10
(1) Construction of heterologous host expression system of Escherichia coli for styAB from Rhodococcus sp. ST-5 and ST-10 and expression and purification of recombinant enzyme
In order to express the styAB gene derived from both Rhodococcus sp. ST-5 and ST-10 strains in Escherichia coli, a plasmid for expression was constructed according to the following procedure. E. coli BL21 (DE3) was used for expression.
1. Oligonucleotide primers (Table 3) specific for the terminal sequences of styAB of ST-5 and ST-10 were prepared, and PCR was performed on ST-5 and ST-10 genomic DNA. At this time, restriction enzyme sites (Bam HI, Hind III) were added to the N-terminal and C-terminal.
2. The amplified fragment of about 1.8 kbp was cloned into pGEM-T Easy vector (plomega) and the internal sequence was confirmed.
3. The styAB fragment was excised by Bam HI and Hind III treatment and purified by agarose gel electrophoresis. The purified styAB fragment was ligated to pET-21 (b) pretreated with the same restriction enzyme and introduced into E. coli DH5α.
4. Transformants were selected on antibiotic plates. The plasmid was extracted and purified, and introduced into E. coli BL21 (DE3) by electroporation. Furthermore, a chaperone expression vector pG-KJE8 was introduced in an overlapping manner in order to increase the efficiency of recombinant protein expression.
5. Transformants were selected on antibiotic plates and used for subsequent expression experiments.
A construct for expressing styA alone was also prepared by the same method as described above.
The primer sequences used for the construction of the expression plasmid are shown in Table 3, and the constructed plasmid is shown in FIG.

Analysis of SMO molecular weight by SDS-PAGE was performed under the following conditions.
1. StyA sample containing purified histidine tag was dissolved in 2x SDS-PAGE sample buffer (100 mM Tris-HCl, 20% glycerol, 4% SDS, 0.05% bromophenol blue).
2. The above sample corresponding to a protein amount of 5 μg was applied to a 10% polyacrylamide gel.
3. Run at 150 V, 40 mA for 100 minutes.
4. The electrophoresed gel was immersed in an SDS-PAGE staining solution (50% methanol, 10% acetic acid, 0.25% Coomassie blue) and stained while shaking for 40 minutes.
5. The stained gel was immersed in a decoloring solution (30% methanol, 10% acetic acid), and decolorization was performed while appropriately replacing the decolorizing solution.
6. From the mobility of the obtained band, the molecular weight of purified StyA was estimated to be about 49.0 kDa. For calculating the molecular weight, a molecular weight marker (Prestained APRO-Marker) that was run simultaneously with the sample was used as an index.

The SMO molecular weight analysis by gel filtration was performed under the following conditions.
1. Shimadzu LC-10 HPLC system was used as the analytical system, and Superdex 200 10/300 GL manufactured by GE Healthcare was used as the analytical column.
2. Separation was performed at a flow rate of 0.4 ml / min using 100 mM KPB, 200 mM NaCl pH 7.0 as the mobile phase.
3. 100 μl of purified StyA sample containing histidine tag (equivalent to 500 μg protein) was applied as an analytical sample and eluted as a single peak at 35.3 min.
4. Similarly, a standard sample (Oriental Yeast MW-Marker (HPLC)) was analyzed.
5. The molecular weight and elution time of the standard sample are shown below.
Yeast glutamate dehydrogenase 290 kDa 26.9 min
Porcine myocardial lactate dehydrogenase 142 kDa 31.3 min
Yeast enolase 67 kDa 34.3 min
Yeast myokinase 32 kDa 39.1 min
Horse heart cytochrome c 12.4 kDa 43.0 min
6. A calibration curve was prepared using the elution time and molecular weight of each protein in the above standard sample. As a result of plotting the elution time 35.3 min of purified StyA against the obtained calibration curve, it was estimated that the molecular weight of StyA corresponds to 59.2 kDa.

Recombinant StyAB was expressed and purified as follows.
1. The transformant containing the expression plasmid was cultured in 4 ml of LB medium (50 μg / ml ampicillin, 20 μg / ml chloramphenicol) at 37 ° C. and 170 rpm.
2. Inoculate 1 ml of the preculture into 100 ml LB medium (50 μg / ml ampicillin, 20 μg / ml chloramphenicol, 0.5 mg / ml arabinose, 5 ng / ml tetracycline), and culture at 37 ° C and 250 rpm did.
3. When OD ₆₀₀ reached 0.5, IPTG was added to a final concentration of 0.1 mM and induced at 18 ° C., 250 rpm for 24 hours.
4. The cells were collected by centrifugation (18000 rpm, 4 ° C., 10 min), suspended and washed in 50 mM KPB (pH 7.0), 10% glycerol.
5. The cells were collected again by centrifugation and resuspended in Ni-Sepharose column Binding Buffer (50 mM KPB, 10% glycerol, 500 mM NaCl, 10 mM imidazole, pH 7.5).
6. The cells were disrupted by ultrasonic disruption, and the supernatant was collected by centrifugation (18,000 rpm, 4 ° C., 20 min × 2 times).
7. Ni-Sepharose column The supernatant of the cell disruption solution was applied to a Ni-Sepharose High performance column equilibrated with Binding Buffer to adsorb the target recombinant protein.
8. After washing the column with 10 times the column volume of Binding Buffer, the target protein adsorbed with Elution Buffer (50 mM KPB, 10% glycerol, 500 mM NaCl, 500 mM imidazole, pH 7.5) was eluted.
9. The resulting purified protein solution was dialyzed and replaced with 50 mM KPB, 10% glycerol, pH 7.0.
10. The dialyzed protein solution was finally purified using various ion exchange chromatography (DEAE-Sepharose, Resource Q, MonoQ) to obtain a purified sample of the target protein.

(2) SMO activity measurement
ST-5 and ST-10-derived SMO activity was measured using a column-purified recombinant enzyme. Each purified enzyme, substrate, and coenzyme were added to a 50 mM MES buffer at the following concentrations to obtain a reaction mixture.
Reaction mixture composition
50 mM MES Buffer (pH 7.0)
20 mM sodium formate
100μM NADH
200μM FAD
1 U formate dehydrogenase
10 U catalase
5 mM substrate (react with 10 mM substrate during substrate conversion reaction)
StyA 150μl (In the substrate conversion reaction, use the cell disruption supernatant of StyAB co-expressing cells)
StyB 100μl
total 1000μl
This reaction mixture was incubated at 30 ° C. and 2,500 rpm for 30 minutes. After completion of the reaction, 500 μl of ethyl acetate was added to the reaction mixture and vortexed for 5 minutes. After centrifugation at 15,000 rpm for 5 minutes, the separated ethyl acetate phase was transferred to a new tube. Again 500 μl of ethyl acetate was added to the aqueous phase, vortexed for 5 minutes, and then the ethyl acetate phase was recovered by centrifugation again. An appropriate amount of sodium sulfate was added to a total of 1000 μl of the collected ethyl acetate phase, followed by vortexing for 2 minutes for dehydration. The dehydrated extracted sample was analyzed using a Hewllet Packerd HP6890 gas chromatography system.
1 U was defined as the enzyme activity that converts 1 μmol of substrate into product per minute.

(3) Gas Chromatography analysis conditions
ST-5 and ST-10-derived SMO reaction products were measured using gas chromatography. The reaction product reacted under the above reaction conditions and extracted with ethyl acetate was analyzed using a Hewllet Packard HP6890 gas chromatography system. 1 μl of the sample extracted with ethyl acetate was injected into a CP-Cyclodextrin-b-2,3,6-M-19-25 capillary column (25 m, inner diameter 0.25 mm) manufactured by Varian. The carrier gas (99.999% high purity helium gas) flow rate was 2.8 ml / min, the split ratio was 50, and the linear flow rate was 46 cm / s. Detection was performed using a FID (flame ionization detector). The vaporization chamber temperature was 250 ° C and the detector temperature was 250 ° C.
The temperature program of the column oven was performed using a constant temperature program. Table 4 shows the column oven temperature and the detection time of each substance for each analysis sample.
The identification and quantification of each substance was determined by analyzing a sample prepared with each standard compound at a concentration of 1 mM, 5 mM, and 10 mM under the same conditions, and creating a calibration curve from the detection time and peak area.

(4) Substrate specificity of styA derived from Rhodococcus sp. ST-5 and ST-10
ST-5 and ST-10-derived SMO showed epoxidation activity against various substrates including styrene (Table 5). The substrate specificity was approximately twice that of 4-fluorostyrene when styrene was 100. Also, the enantiomeric excess (ee) of the product is slightly lower for styrene, 4-chlorostyrene, 4-bromostyrene, 4-fluorostyrene, but for 2-chlorostyrene and 3-chlorostyrene. The value was 99.9% or more, and the absolute configuration was (S) -form.

反応産物の立体選択性については、styrene oxide および3-chloro styrene oxide 以外は産物の標品が市販されていないか、ラセミ体しかないため、S-体であると推測されるが確認はできていない。

As for the stereoselectivity of the reaction product, it is estimated that it is S-form because the product preparations other than styrene oxide and 3-chloro styrene oxide are not commercially available or only racemic. Absent.

  The present invention provides a styrene monooxygenase and a polynucleotide encoding the enzyme. By using the styrene monooxygenase of the present invention, styrene monooxygenase can be produced efficiently.

Claims (9)

The polynucleotide according to any one of (a) to (c) and (e) below:
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 or 3;
(B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(C) the amino acid sequence of SEQ ID NO: 2 or 4, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and described in (1) and (2) below polynucleotides encoding styrene monooxygenase having the following physicochemical properties; Contact and <br/> (e) SEQ ID NO: 2 or 4 having the amino acid sequence identity of 90% or more of the described and following (1 And a polynucleotide encoding a styrene monooxygenase having the physicochemical properties described in (2);
(1) Action
FADH ₂ (reduced FAD) is used as a coenzyme to act on styrene and 3-chlorostyrene to produce optically active styrene oxide and optically active 3-chlorostyrene oxide, respectively.
(2) Molecular weight This is a monomer enzyme having a molecular weight of 59,000 including histidine in gel filtration, a molecular weight of 49,000 by SDS-PAGE, and an estimated molecular weight of 47,000 from the amino acid composition. The polynucleotide according to claim 1, which is derived from a microorganism belonging to the genus Rhodococcus. The polynucleotide according to claim 2, wherein the microorganism belonging to the genus Rhodococcus is Rhodococcus opacus ST-5 strain or Rhodococcus erythropolis ST-10 strain. A protein encoded by the polynucleotide according to any one of claims 1 to 3. A vector comprising the polynucleotide according to any one of claims 1 to 3. A transformed cell into which the polynucleotide according to any one of claims 1 to 3 or the vector according to claim 5 has been introduced. The polynucleotide according to any one of claims 1 to 3, comprising a step of culturing the transformed cell according to claim 6 and recovering a protein encoded by the polynucleotide according to claim 1 from the culture. A method for producing a protein encoded by An optically active styrene oxide and / or an oxide of an optically active styrene analog, comprising the steps of causing the protein according to claim 4 to act on styrene and / or an analog thereof, and recovering a produced compound. Production method. A method for decomposing styrene and / or an analogue thereof, comprising the step of causing the protein according to claim 4 to act on styrene and / or an analogue thereof.

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