JP2006314224A - Method for producing aromatic monohydroxy compound, and aromatic monohydroxy compound obtained thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To readily and efficiently obtain an aromatic monohydroxy compound from an aromatic compound. <P>SOLUTION: The aromatic monohydroxy compound, e.g. monohydroxydibenzothiophene is produced by bringing a microorganism in which the enzyme activity of a polycyclic aromatic compound-splitting enzyme (bphB) is inhibited by gene-breaking, expression inhibition, use of inhibitor, or the like, or a cell content of the microorganism, e.g. the microorganism of the genus Sphingomonas into contact with the aromatic compound, e.g. dibenzothiophene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing an aromatic monohydroxy body and an aromatic monohydroxy body obtained thereby.

  A compound in which a hydroxyl group is added to a polycyclic aromatic compound is a substance that can be used as a raw material for various products such as petroleum products, herbicides, and pharmaceuticals. Many studies have been conducted for process production. The production of industrial products using bioprocesses is excellent in terms of cost, and is expected as an alternative to conventional chemical synthesis as an environmentally friendly technology.

Regarding the hydroxylation of polycyclic aromatic compounds (PAH) by monooxygenase, Mori et al., P450 monoxygenase of white rot fungi (Phlebia lindtneri) is position specific of dibenzodioxin (DBD), dibenzofuran (DBF), biphenyl (BPH) Have been reported to be responsible for basic hydroxylation (Non-patent Document 1). In addition, in filamentous fungi and yeast, there are some reports on the degradation of polycyclic aromatic compounds via hydroxy compounds (for example, Non-Patent Document 2).
However, white rot fungi are only responsible for hydroxylation of the substrate, and the decomposition does not proceed thereafter.
On the other hand, it has been clarified that monocyclic benzene and toluene have a ring-cleavage decomposition pathway via a monohydroxy compound (Non-patent Document 3). It has also been reported that dihydrodiols can be obtained using various aromatic ring dioxygenases using microorganisms (Patent Documents 1 to 4).
Appl.Microbiol.Biotechnol., 60, 200-205, (2002) Appl.Environ.Microbiol., 67 (4), 1551-1557, (2001) Aldrichimia Acta, Vol. 32, (2), 35-62, (1999) JP-A-5-130875 JP-A-6-181789 Japanese Patent Laid-Open No. 2003-269 JP 2003-93087 A

If the aromatic compound can be directly monohydroxylated, a monohydroxy compound can be efficiently obtained with few steps.
Accordingly, an object of the present invention is to obtain an aromatic compound monohydroxy form simply and efficiently from an aromatic compound.

The method for producing an aromatic monohydroxy product of the present invention is characterized in that an aromatic compound is contacted with a microorganism in which the enzyme activity of a polycyclic aromatic compound-degrading enzyme (bphB) is suppressed or the cell contents of the microorganism. Yes.
Here, the suppression of bphB enzyme activity is preferably performed by at least one selected from the group consisting of suppression of bphB gene expression and gene disruption and use of bphB activity inhibitors.
The aromatic monohydroxy body of the present invention is obtained by the above production method.

  In the present invention, since a microorganism in which bphB enzyme activity is suppressed or the cell contents of the microorganism is used, the degradation pathway of the aromatic compound becomes incomplete. As a result, a monohydroxy form is generated instead of a dihydroxy form.

  According to the present invention, an aromatic monohydroxy product can be easily and efficiently obtained from an aromatic compound.

The method for producing an aromatic monohydroxy body of the present invention includes contacting an aromatic compound with a microorganism in which the enzyme activity of bphB is suppressed or the cell contents of the microorganism.
In general, only the diol-mediated pathway is known as the degradation pathway of polyaromatic compounds. For example, only the so-called Kodama pathway shown in FIG. 1 has been scientifically proven as the degradation pathway of dibenzothiophene. Absent.
In the production method of the present invention, a microorganism in which the enzyme activity of bphB involved in this pathway is suppressed or the cell contents of the microorganism are used. As a result, the degradation pathway of the aromatic compound becomes incomplete, and the details of the mechanism of synthesis are unknown. However, the aromatic compound is directly hydroxylated without by-production of the dihydroxy compound, and it can be conveniently used in a one-step reaction. And a monohydroxy body can be obtained efficiently. The monohydroxy form obtained by this method has a high purity as compared with, for example, a route using dioxygenase derived from other microorganisms.

  bphB is an enzyme that exists in microorganisms and is capable of degrading polycyclic aromatic compounds having two or more aromatic rings and aromatic rings containing heteroatoms, and bphB protein is known as a so-called short chain dehydrogenase. Yes. The crystal structure of the bphB protein was first determined for bphB from Burkholderia sp. LB400 by Hulsmeyer et al. (Protein Sci., 7 (6), 1286-93, (1998).). The Burkholderia sp. LB400 strain has substrate specificity for various polychlorinated biphenyls (BPH), and degrades and assimilates biphenyl through the following ring-cleavage-type degradation pathway.

BphB of LB400 forms a tetramer and uses NAD ⁺ as a coenzyme. The mechanism of action of bphB in LB400 is shown below. bphB is considered to dehydrogenate dihydrodiol by the following two-step reaction mechanism.

(A) Tyr155 is deprotonated. (B) Deprotonated Tyr forms a hydrogen bond with the 3-hydroxyl group of the substrate. Next, Tyr155 takes in the proton of the 3-hydroxyl group of BPDD (cis- (2R, 3S) -dihydroxy-1-phenylcyclohexa-4,6-diene), releases 3-H, and the substrate is a coenzyme. It binds to NAD ⁺ which is (C) NAD (H) incorporating hydrogen is liberated from the substrate, and α-hydroxycyclohexa-2,4-diene, which is a reaction intermediate, is generated. Due to keto-enol tautomerism, this material undergoes rearrangement of the benzene ring in the sub-reaction, producing 2,3-dihydroxy BPH (DHBP).

  The nucleic acid sequence and amino acid sequence of this bphB are already known as SEQ ID NOS: 1 and 2, but bphB in the present invention is not limited to the sequences of SEQ ID NOS: 1 and 2 as long as they have similar functions. , One or more of the nucleic acids or amino acids may be deleted, substituted or added.

  In the present invention, the enzyme activity of bphB may be suppressed, and the enzyme activity may be suppressed by at least selected from the group consisting of suppression of bphB gene expression and gene disruption and use of a bphB activity inhibitor. One is preferred.

The gene region to be destroyed may be a disruption to such an extent that the function of bphB can be destroyed, and it does not have to be the entire bphB sequence. For example, partial deletion of the coding region and catalytic active region sequences and destruction of the promoter sequence can be mentioned. Among these, from the viewpoint of efficiently destroying the function of bphB, destruction of the catalytic active region is preferable .
Gene disruption and gene expression suppression can be appropriately selected and easily used by those skilled in the art as long as they are methods commonly performed in this field. For example, examples of gene disruption include random gene disruption using a transposon and region-designated disruption by homologous recombination. Examples of gene expression suppression include antisense and RNAi. The application of these techniques to such a technique can be appropriately used by those skilled in the art since the target sequence is clear.

  Moreover, even if bphB gene expression is performed, the same effect as described above can be obtained as long as the enzyme activity can be inhibited. For this reason, the inhibitor of the enzyme activity of bphB can also be used without particular limitation as long as it is a compound having this function.

  In addition, as other methods for suppressing the enzyme activity of bphB, there can be mentioned the destruction and suppression of expression of other metabolic enzyme genes corresponding to bphB. As such metabolic enzyme genes, dihydrodiol dehydrogenase can be mentioned. Can do. The enzyme activity of bphB can also be suppressed by disrupting this metabolic enzyme gene.

In the present invention, a microorganism in which bphB enzyme activity is suppressed or a cell content of the microorganism is used.
The microorganism that can be used in the present invention is not particularly limited as long as it is a microorganism having an aromatic compound resolving ability, but is selected from the group consisting of gram-negative bacteria and gram-positive bacteria from the viewpoint of product production and recovery efficiency. At least one is preferable, and among them, industrial value and convenience are at least one selected from the group consisting of the genus Sphingomonas, Pseudomonas, and Rhodococcus, which are industrially and widely used as research objects. It is further preferable from the viewpoint of safety. These microorganisms can be appropriately selected according to the type of target monohydroxy compound and the type of aromatic compound serving as a substrate. Of these microorganisms, the Sphingomonas genus such as Sphingomonas paucimobilis is particularly preferable from the viewpoint of efficiently producing the compound of the present invention.

The cell content of the microorganism is not particularly limited as long as the cell content is extracted according to a normal method in which intracellular enzyme activity is not lost, and lysate using a lytic enzyme such as lysozyme, ultrasonic wave Examples thereof include pulverized products using a crusher, a French press, a homogenizer, or the like.
Here, from the viewpoint of easily recovering the produced monohydroxy form, it is preferable to use the microorganism as it is.

  The microorganism used in the present invention may be the same as the natural one except that the enzyme activity of bphB is suppressed, and the monohydroxy form in the present invention is converted using other biological functions in this microorganism. Can be generated. Examples of other functions in the microorganism include naturally occurring enzyme activity and the like, but may be modified so as not to impair the effects of the present invention. Such modifications can include improvements in other enzyme activity efficiencies and growth rates. These modifications can be appropriately selected by those skilled in the art and can be easily implemented.

  In order to obtain a monohydroxy compound in the present invention, the above-described microorganism or the cell content thereof may be brought into contact with an aromatic compound.

The aromatic compound to be used is not particularly limited as long as it is an aromatic compound which is the basis of the target monohydroxy compound and bphB is related to the decomposition pathway.
Here, the aromatic compound means one having two or more, for example, a tricyclic or tetracyclic condensed or non-condensed aromatic ring in the molecule, and a polycyclic aromatic compound and aromatic from an industrial viewpoint. It is preferably selected from the group consisting of amino acids. This aromatic compound has one or more side chains each composed of a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom, a lower saturated hydrocarbon group or an alicyclic hydrocarbon group, in the molecule. May be. The lower saturated hydrocarbon group includes an alkyl group having 1 to 6 carbon atoms and an alkenyl group such as methyl, ethyl, propyl, butyl, pentyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2- Examples thereof include methyl-2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl and the like. Examples of the lower alicyclic hydrocarbon group include a cycloalkyl group having 3 to 8 carbon atoms and a cycloalkenyl group such as cyclopropyl, cyclopentyl, and cyclopentene. The aromatic ring contained in the compound is not particularly limited as long as it is cyclic, and preferred examples include 5-membered rings and 6-membered rings to 8-membered rings. As such an aromatic compound, those having 5 to 50 carbon atoms, preferably 6 to 40 carbon atoms, are applicable because they can be produced efficiently from an industrial point of view. Biphenyl and dibenzofuran are used as polycyclic aromatic compounds. Examples of aromatic amino acids such as dibenzodioxin and dibenzothiophene include phenylalanine, tryptophan, tyrosine, and histidine.

  In the production method of the present invention, the aromatic compound oxidoreductase involved in the pre-stage of bphB in the synthesis pathway is not particularly limited. For this reason, even if it is a system in which any oxidoreductase is involved, a monohydroxy body can be easily obtained as described above.

  As conditions for contacting the aromatic compound with the microorganism or the cell contents of the microorganism, normal conditions necessary for assimilation of the compound by the microorganism to be used may be applied as they are, and those skilled in the art can use the microorganism to be used. It can be appropriately selected depending on the species.

Since the aromatic monohydroxy substance produced | generated by the said contact process exists in a reaction product normally as a single compound, extraction of an aromatic monohydroxy substance can be performed easily.
The extraction method used here may be any method as long as it is a method used for purification of general low molecular weight compounds. For example, the product in the culture filtrate is extracted with a water-immiscible organic solvent, such as ethyl acetate, or the product in the cell is filtered, centrifuged, etc. Examples thereof include a method of collecting with ethanol, acetone and the like, and a countercurrent distribution method using an appropriate solvent. The culture as it is can be subjected to the above extraction operation without separating the cells. In addition, suitable adsorbents such as silica gel, activated carbon, “Diaion HP-20” (Mitsubishi Kasei) can be used for product-containing products that are already liquid, such as culture filtrates and extracts obtained by extraction operations. The product of interest can be adsorbed using a suitable solvent and then eluted with a suitable solvent. If the product solution thus obtained is concentrated to dryness under reduced pressure, a crude product preparation is obtained. In order to further purify the product crude sample, the extraction method and the adsorption method described above may be combined with gel filtration, high performance liquid chromatography, etc., as necessary, and performed as many times as necessary. For example, column chromatography using an adsorbent such as silica gel, gel filtration agent such as “Sephadex LH-20” (Pharmacia), high-performance liquid chromatography using “YMC pack” (Yamamura Kagaku) and the like A countercurrent distribution method can be implemented in combination as appropriate.

The monohydroxy form obtained by the present invention can be identified by ¹ H NMR and ¹³ C NMR spectrum analysis, MS spectrum analysis and the like. The obtained aromatic compound monohydroxy compound is used as a functional substance that is the center of various fields, for example, a starting material for pharmaceuticals or a starting material for conductive polymers. A hydroxy body can be provided efficiently.

  Thus, according to the manufacturing method of the aromatic compound monohydroxy body of this invention, various aromatic compound monohydroxy bodies can be obtained in a short time by one-step reaction. Moreover, since an aromatic compound can be efficiently assimilated into a monohydroxy compound, it can be effectively applied to environmental improvement.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

[Example 1]
Production of dibenzothiophene (DBT) and various aromatic monohydroxy compounds by disruption of bphB gene in TZS-7 strain (1) Gene disruption in TZS-7 strain It is known to have polycyclic aromatic compound resolution, that is, bphB gene Homologous recombination method, which is one of the standard methods for gene disruption of microorganisms, to the bphB gene of Sphingomonas paucimobulis TZS-7 strain (Journal of Bioscience and Bioengineering 88: 293-299 (1999)) Was used to introduce a kanamycin resistance gene. Specifically, a DNA fragment in which the drug resistance gene kanamycin resistance gene is inserted between the 27th and 28th bases of the ORF (see FIG. 2) is prepared and introduced into the TZS-7 strain by electroporation. I got a substitute. By directly analyzing the base sequence, it was confirmed that the bphB gene was destroyed. This strain was designated as Tn399.

(2) Determination method of base sequence After inoculating the Tn399 strain obtained above in 4 ml of LB medium and shaking culture at 37 ° C overnight, the culture solution was transferred to GenElute Plasmid Mini-Prep Kit (Sigma Co., Ltd.). The plasmid was prepared. This plasmid preparation solution or a PCR reaction product using the plasmid as a template was used.
LA Taq polymerase (Takara Shuzo Co., Ltd.) was used to prepare a sequence template by PCR. In accordance with the instructions attached to the LA Taq polymerase, about 5 ng of the plasmid dissolved in LA Taq polymerase 0.5 U and sterilized water, 5′-M13 primer (M13-20 (5′-GTAAAACGACGGCCAGT-3 ′) : SEQ ID NO: 3) and 3′-M13 primer (RP (5′-GGAAACAGCTATGACCATG-3 ′): SEQ ID NO: 4) were each used at a final concentration of 0.2 μM to prepare a total volume of 50 μl. The PCR reaction was first heat-denatured at 94 ° C. for 3 minutes, and then subjected to a total of 30 cycles of 94 ° C. for 3 minutes and 68 ° C. for 5 minutes. Subsequently, the PCR reaction product was corrected and extended at 70 ° C. for 10 minutes, and finally stored at 4 ° C.
The obtained PCR product was subjected to UltraClean PCR Clean-up DNA Purification Kit (manufactured by Takara Shuzo Co., Ltd.) to remove unnecessary primers, unreacted dNTPs, etc., and used as a template for sequencing.

A cycle sequence reaction was performed using the template prepared as described above and ABI PRISM Big Dye ^™ Terminator Cycle Sequencing Ready Reaction ver. 2 (manufactured by ABI).
First, in a 0.2 ml micro test tube, 4 μl of the Big Dye ver.2 included in the plasmid preparation or PCR reaction product and the kit, and EZ :: TN ^TM <KAN-2> Tnp Transposome ^TM Kit 1 μl of KAN-2 FP-1 Forward Primer or KAN-2 RP-1 Reverse Primer (0.2 pmol / μl) was added to prepare a total volume of 10 μl with ultrapure water.
These samples were then subjected to a PCR reaction. First, after heat denaturation at 96 ° C. for 1 minute, one cycle was 96 ° C., 30 seconds, 45 ° C., 5 seconds, 60 ° C., 4 minutes, 25 cycles were performed, and the reaction was stored at 4 ° C. after completion of the reaction. Thereafter, 10 μl of sterilized water, 2 μl of 3.0 M sodium acetate and 99.5% ethanol were added to the sequence reaction solution, mixed, and allowed to stand at room temperature for 10 minutes. Subsequently, the mixture was centrifuged at 15,000 rpm, 18 ° C. for 30 minutes, the precipitate was washed with 70% ethanol, and the DNA pellet was dried with an evaporator. 15 μl of PE TSR was added to the dried DNA pellet, dissolved sufficiently, incubated at 95 ° C. for 2 minutes, and then rapidly cooled in ice to prepare a sample.

(3) Computer analysis of base sequence The sample prepared as described above was subjected to Genetic Analyzer 310 manufactured by ABI, and the base sequence of the inserted fragment was determined.
The obtained nucleotide sequence data was analyzed using GENETYX-MAC (Ver.11.0), a computer program manufactured by SDC Software Development. Blast was used for homology analysis of DNA and amino acids.

(4) Alignment of amino acid sequence of bphB A comparison was made between the protein obtained by converting bphB in the TZS-7 strain into an amino acid sequence and the protein encoded by the highly homologous gene. The results are shown in FIG.
As shown in FIG. 3, bphB (putative dihydrodiol dehydrogenase) of Sphingomonas aromaticivorans F199 strain and 89.5%, ORF4 (putative 1,2-dihydrodiol dehydrogenase) and 75.6% of Sphingomonas sp. BN6 strain, Pseudomonas sp. 44.0% homology was confirmed with bphB (cis-2,3-dihydrobiphenyl-2,3-dehydrogenase) of the KKS 102 strain. These results suggested that the gene disrupted by the transposon was bphB encoding dihydrodiol dehydrogenase. As a result of the Blast domain search of bphB in TZS-7, bphB was classified into the short-chain alcohol dehydrogenase family (SDR family). The SDR family is conserved from microorganisms to humans, but its direct reaction has not been investigated in detail.

A feature common to the SDR family is known to have a GlyXXXGlyXGly motif, which is considered to be a binding site for the coenzymes NAD (H) and NADP (H), and a TyrXXXLys structure, which is a catalytically active site. Yes. These motifs were also confirmed from the bphB sequence of the TZS-7 strain.
Although bphB Lys159 in LB400 is not directly involved in this reaction mechanism, Lys159 can polarize Tyr155 and keep the acidity index (pKa value) of the hydroxyl group low by electrostatic interaction. This effect is thought to promote the deprotonation of Tyr155 and consequently promote the enzyme catalyst.
From the three-dimensional structure analysis of bphB in LB400, it was considered that three amino acids Ser142, Tyr155, and Lys159 are catalytically active groups. Vadai et al. (Biochemistry, 39 (17), 5028-34, (2000)) proved this possibility further experimentally.

  Vadadi et al. Are biphenyl (BPH), a polychlorinated biphenyl degrading bacterium, and bphB (cis-2,3-Dihydro-2,3-dihydroxybiphenyl Dehydrogenase) of Comamonas testosteroni B-356, which is highly homologous to bphB of LB400. Ser145, Tyr158, and Lys161 are each substituted with an amino acid by site-directed mutagenesis to produce mutant bphB, and its dynamics are analyzed. As a result, it was suggested that these amino acids are catalytically active groups. They called these three amino acids “catalytic triad” (active central amino acid) and claimed to be an essential amino acid for bphB catalytic activity. This “catalytic triad” was also preserved in bphB of TZS-7 used in this example (FIG. 3).

[Example 2]
Production of monohydroxy DBT by disruption of bphB (1) Test strain TZS-7 strain and DBT degradation-related enzyme gene (bphB) disruption strain (Tn399) in TZS-7 strain
(2) Medium and culture conditions The degradation rate of each substrate was calculated by resting cell reaction. First, the strain stored at 4 ° C. on the NB agar medium is scraped off, inoculated into a medium test tube containing 5 ml of NB medium, and cultured at 30 ° C. until reaching the stationary phase (OD ₆₆₀ = 1.5). did. Thereafter, 200 μl of the culture solution and 20 ppm of toluene for induction of PAH degrading enzyme were added to a 500 ml Erlenmeyer flask containing 100 ml of NB medium, and cultured at 30 ° C. with shaking. In addition, toluene diluted to 100 μl with acetone was added.
When the OD reached 1.0, 20 ppm of toluene was added again to induce again. When the OD reached 1.3, the culture was centrifuged at 4,500 rpm for 20 minutes to recover the cells. Next, 10 ml of 0.1 M phosphate-potassium buffer solution (pH 7.0) was added to the cells, and the mixture was thoroughly stirred by vortexing, and then centrifuged again at 4,500 rpm for 20 minutes to collect the cells. This operation was repeated twice. After washing, a resting cell suspension was prepared with the same buffer so that the OD ₆₆₀ was 10. When the suspension was stored, it was freeze-stocked at −80 ° C., and when used, it was melted in ice-cold water.

  500 μl of resting cells prepared as described above were added to a small test tube cooled on ice, and 10 μl of 5 g / l DBT / DMF solution or 4,6-DMDBT / DMF solution (final concentration 100 ppm) was added thereto. ), And a resting cell reaction was carried out at 30 ° C. and 600 rpm. After the reaction, the mixture was acidified by adding 50 μl of 6N HCl, and then ethyl acetate equivalent to the reaction solution was added and stirred well by vortexing. After leaving still for 5 minutes, the water layer and the organic layer were separated by centrifugation, and the substrate was extracted.

(3) Reagent DBT used was dissolved in acetone at a concentration of 10 g / l (unsterilized). Unless otherwise specified, other reagents were sterilized by filter or autoclaved. The DNA-related enzyme used for PCR, the buffer reagent used for the preparation of resting cells, etc. were manufactured by Takara Shuzo Co., Ltd., Toyobo Co., Ltd., or Invitrogen.
(4) Analysis conditions GC used was Shimadzu GC-2010. The GC system includes a flame ionization detector (FID) and a capillary column (DB-1, J & W Scietific). The column temperature was 260 ° C., and the injector and detector temperatures were 300 ° C., respectively.
The degradation product in the resting cell reaction of Tn399 was used as a sample for GC / MS.
The structure analysis of the DBT degradation product was determined by mass spectrometry gas chromatography (Gas chromatography / mass spectrometry, GC / MS). The GC / MS system (Thermo Quest) was equipped with a capillary column, and the column oven was heated from 150 ° C. to 275 ° C. (gradient, 10 ° C./min). For the analysis of mass spectrum, spectrum search software “Xcalibur” attached to GC / MS was used.

(4) Comparison of growth curves of TZS-7 and recombinant strain Tn399 Growth curves of TZS-7 (square) and Tn399 (diamond) are shown in FIG. In the NB medium, no difference in growth was observed between the wild strain and the mutant strain, and it was revealed that the gene that had been disrupted by transposon at Tn399 did not affect the growth. Therefore, it was clear that the resting cell preparation method established in TZS-7 can be directly applied to Tn399.
(5) GC / MS Analysis of DBT Degradation Product in Tn399 GC GC in the GC / MS analysis and a mass spectrum of a peak peculiar to Tn399 are shown in FIG. From the mass spectrum, this compound was estimated to have a molecular weight (M ⁺ ) of 200, and it was estimated that an oxygen atom was added to any position of DBT (MW = 184). However, it was considered to be a substance different from DBT sulfoxide from the retention time of the chromatogram, and the mass spectrum of peak 1 suggested that the compound accumulated in Tn399 was hydroxy DBT.

[Example 3]
Analysis of monohydroxy DBT of Tn399 by ¹ H-NMR (1) Preparation of resting cells and reaction of resting cells For the method of preparing resting cells of TZS-7 and Tn399, the same method as described in Example 2 was used. Using. In the resting cell reaction, 50 ml of resting cells were added to a 300 ml feathered Erlenmeyer flask, 5 mg (final concentration 100 ppm) of DBT dissolved in N, N-dimethylformamide (DMF) was added thereto, and 30 rpm 200 rpm. For 24 hours. In order to minimize the influence of the organic solvent on the cells, the amount of DMF used for dissolving DBT was dissolved at the minimum amount at which DBT was solubilized visually, and was prepared at the time of use.

(2) Extraction of DBT degradation product The reaction solution of (1) was centrifuged at 4,500 rpm for 40 minutes to remove cells and undegraded substrate. The supernatant was transferred to a thick test tube and acidified by adding 1/10 of 6N HCl to the sample, and then the same amount of ethyl acetate as the sample was added, and the mixture was stirred well by vortexing. After leaving still for 5 minutes, it moved to the glass centrifuge tube, and centrifuged at 4,500 rpm for 20 minutes, and isolate | separated the water layer and the organic layer. The organic layer was collected and the DBT degradation product was extracted. This operation was repeated twice.
The extracted samples were put together in a 100 ml glass conical flask with a lid, and an appropriate amount of anhydrous sodium sulfate was added and allowed to stand overnight to dehydrate the sample. Subsequently, the sample was subjected to a rotary evaporator to concentrate the decomposition products.

(3) Isolation of the target compound by silica gel column chromatography The weight of the prepared sample was measured, and the sample was dissolved in acetone so as to be 1,000 ppm. The dissolved sample was subjected to TLC.
First, a 5 × 20 cm TLC plate silica gel 60F ₂₅₄ (MERCK) was cut into 5 × 3 cm pieces by glass cutting and used for the following TLC operations. A 20% acetone-chloroform solution was added to the development layer, a TLC plate spotted with the sample and the substrate was added thereto, the sample was developed, and the compounds contained in the sample were predicted. Next, the system was developed sequentially into 100% hexane, 5% diethyl ether-hexane solution, and 10% diethyl ether-hexane solution, and a solvent system capable of isolating the target compound was examined. In addition, about the elution power of the mixed solvent system in silica gel, introductory chromatography 1st edition, Tokyo Chemical Dojin, (1971) pp.35.

Silica gel column chromatography was performed based on the TLC results. First, add absorbent cotton or silica wool to a disposable Pasteur pipette (manufactured by IWAKI), and add silica gel to the upper layer 10 times the sample weight (however, at least 500 mg of silica gel) to create a simple column did.
A column chromatography solvent system was constructed based on the TLC results. First, the sample was transferred in a solvent system (100% hexane in this case) to be developed, and the silica gel was washed with the same solvent. Washing was performed until it was confirmed that the air in the silica gel was completely removed visually.

The sample was passed through the column prepared as described above, and then eluted sequentially using 5 ml each of hexane 100%, 5% diethyl ether-hexane solution, 10% diethyl ether-hexane solution, and 20% acetone-chloroform solution. It was. The eluted sample was collected in a glass centrifuge tube (manufactured by Sanyo Co., Ltd.) for each solvent system.
The collected sample was again subjected to TLC with a 20% acetone-chloroform solution to confirm whether the target compound was isolated. In addition to TLC, GC and GC / MS also detected compounds in samples and estimated structures. When the isolation of the target compound was confirmed, the sample was concentrated with a rotary evaporator, the weight was measured, and the sample was stored at 4 ° C. to prepare a sample for ¹ H-NMR.

(4) Isolation of the target compound by silica gel column chromatography When column chromatography was performed in the solvent system described above, the presence of a substance considered to be hydroxy DBT in a system eluted with 20% acetone-chloroform solution It was confirmed. From the GC / MS analysis, only hydroxy DBT was detected (data not shown), and the target compound could be isolated.
(5) Analysis of DBT intermediate metabolite of Tn399 by ¹ H-NMR
^From the analysis of ¹ H-NMR, accumulation of 2-hydroxy DBT was confirmed as shown in FIG. This revealed that the intermediate metabolite extracted with ethyl acetate was 2-hydroxy DBT.
Since strong acidification is likely to cause instability of dihydrodiol, an attempt was made to carry out an analysis by performing neutral extraction without adding 6N HCl during extraction, but only 2-hydroxy DBT was detected. . Even if the intermediate metabolite accumulated in Tn399 is DBT's dihydrodiol, it is unlikely that all of it will become 2-hydroxyDBT. It is considered that only DBT is generated.
Therefore, according to the present Example, since only 2-hydroxy DBT can be obtained, it was clear that the target monohydroxy body can be obtained directly, without using the purification process from a mixture.

[Example 4]
GC / MS analysis of various aromatic compound degradation products in Tn399 (qualitative)
(1) Experimental method TZS-7 wild type strain and Tn399 strain were used as test strains.
NB medium (Difco) was used for the resting cell preparation of these TZS-7 and Tn399. The culture conditions were both 30 ° C. and 200 rpm.
The substrates used in this experiment are summarized in FIG. Toluene is a special grade manufactured by Wako Pure Chemical Industries, Ltd., BT, 4,6-DMDBT, DBF, DBD, BPH, 1,2-NT, 2,1-NT, 2,3-NT, ANT, PHE, BNT Using. In addition, reagents such as organic solvents used were special grades manufactured by Wako Pure Chemical Industries, Nacalai Tesque Co., Ltd., or equivalents thereof.
The resting cell preparation method, resting cell reaction, and extraction of degradation products of TZS-7 and Tn399 were performed in the same manner as described above.
GC / MS was used for analysis of the extracted sample. The analysis conditions were the same as in Example 3.

(2) GC / MS analysis of monocyclic aromatic compounds (mono-aromatic) degradation products in Tn399 From the GC / MS analysis of the degradation products of toluene and BT in TZS-7 wild strain or Tn399 strain, Formation of a hydroxy compound as in the case of decomposing was not confirmed. From this result, it was considered that, unlike the degradation of DBT, the degradation of toluene and BT by TZS-7 involves a gene different from the DBT degrading enzyme gene.
(3) GC / MS analysis of the degradation product of the bicyclic aromatic compound (di-aromatic) in Tn399 As shown in FIG. 8, an unknown peak (peak 2) that was not found in the wild strain was also confirmed in the DBF degradation product. . The mass spectrum pattern of peak 1 suggested that this compound was hydroxy DBF. Therefore, it was considered that DBF was also metabolized by the same degradation pathway as DBT.
FIG. 9 shows a chromatogram of a DBD metabolite in Tn399. Similarly, a peak peculiar to Tn399 was confirmed in DBD, and accumulation of hydroxy DBD was shown from its mass spectrum.

  FIG. 10 shows a chromatogram of a BPH metabolite in Tn399. In conventional bicyclic aromatic compounds, only one peak was generated with the decomposition of the substrate, but in BPH, two types of peaks specific to Tn399 were confirmed. It was suggested that they were different monohydroxy compounds, 2-hydroxy BPH (peak 4), 4-hydroxy BPH (peak 5). To date, many examples of producing various hydroxy compounds as intermediate metabolites of BPH have been found in eukaryotic microorganisms such as filamentous fungi and yeasts, as in DBF and DBD. For example, Paecilomyces lilacinus, a white rot fungus introduced as a microorganism that produces hydroxy DBD from DBD, has been reported to produce 2-, 3-, 4-hydroxy BPH as BPH degradation products. Using these compounds as intermediate metabolites, hydroxyl groups are sequentially added, and as a final product, a compound in which the benzene ring on one side of BPH is cleaved is generated. However, no example of producing hydroxy BPH in prokaryotic microorganisms has been reported.

  11 and 12 show GC / MS chromatograms and mass spectra of metabolites in Tn399 of 1,2-NT and 2,1-NT, which are representative aromatic compounds of petroleum pollution. In these compounds as well, peaks peculiar to Tn399 were confirmed, suggesting the presence of hydroxy 1,2-NT (FIG. 11, peak 6) and hydroxy 2,1-NT (FIG. 12, peak 7), respectively.

(4) GC / MS analysis of tricyclic aromatic compound (tri-aromatic) degradation product in Tn399 As shown in FIG. 13, a substance (peak 8) that is considered to be a monohydroxy compound was also detected in PHE. The mass spectrum of peak 1 suggested that it was hydroxy PHE. Moreover, as FIG. 14 shows, in ANT, two types of peaks considered to be a monohydroxy compound were confirmed (peak 9 and peak 10). Each mass spectrum was very similar, suggesting that it was a structural analog of any hydroxy ANT.

  The result that bphB has substrate specificity also for tricyclic PHE and ANT indicates that bphB has substrate specificity for a wide range of aromatic compounds from 2 to 3 rings. . It is not clear how bphB metabolizes monohydroxy compounds and whether bphB is really involved in the metabolism of monohydroxyl compounds, but bphB knockout by transposon inhibits the metabolic system of various PAHs. Furthermore, no dihydrodiol was detected from any of the PAH metabolites, and only the monohydroxy compound was detected, indicating that the TZS-7 strain possesses a metabolic system mediated by the hydroxy compound. Results were obtained.

[Example 5]
Measurement of conversion efficiency to hydroxy form by decomposition of various PAHs (quantitative)
As described above, Tn399 is an excellent microorganism capable of adding hydroxyl groups to various PAHs. However, for the purpose of industrial production by bioprocess, it is first important to obtain a large amount of pure PAH-OH. Therefore, it was decided to verify the conversion efficiency of various PAHs by Tn399.
(1) Preparation of resting cells, resting cell reaction, extraction of PAH degradation product The resting cell preparation method and resting cell reaction of Tn399 were performed in the same manner as described above. In order to measure the conversion efficiency of PAH into a hydroxy compound, a calibration curve of hydroxy DBT was used for quantification of the hydroxy compound.
Table 1 shows the conversion efficiency of PAH by Tn399. The highest conversion efficiency was confirmed for 1,2-NT, around 90%. Subsequently, conversion efficiency was high in the order of PHE, DBT, DBD, 2,1-NT, and ANT, and an efficiency of about 30 to 60% was confirmed for each.

  From these, it is clear that according to the present invention, a monohydroxy form of an aromatic compound can be produced efficiently.

It is the figure which showed the digenzothiophene ring cleavage decomposition path | route conventionally known. It is a figure which shows the base sequence of the bphB gene discarded by a transposon in a present Example. It is a figure which shows amino acid sequence alignment of bphB and various microorganisms in TZS-7. In the figure, the arrows indicate the nucleic acid binding motif and the catalytic active site that are also conserved in the bphB gene of TZS-7. TZS-7 bphB aa Seq: bphB of TZS-7 strain, pNL1: bphB of Sphingomonas aromaticivorans F199 strain, SDHD: ORF4 of Sphingomonas sp. BN6 strain, DOXE: DoxE of Pseudomonas putida C18 strain, KKS.BPHS: Pseudomonas sp. Each bphB of the strain is represented. It is a graph which shows the growth curve in the NB culture medium of TZS-7 and Tn399. It is a graph which shows GC / MS analysis of DBT degradation product in Tn399, (A) is a DBT degradation product GC chromatogram of a wild strain, (B) is a DBT degradation product GC chromatogram of Tn399, (C) is detected in Tn399. 2 shows mass spectra of the prepared DBT intermediate metabolite (peak 1). ^{1 is} a ¹ H-NMR spectrum of a DBT decomposition product in Tn399. It is a list of the aromatic compounds used in the Example of this invention. It is a graph which shows GC / MS analysis of the DBF degradation product in Tn399, (A) shows GC chromatogram of DBF degradation product, (B) shows the mass spectrum of the peak 2 in (A). It is a graph which shows GC / MS analysis of the DBD decomposition product in Tn399, (A) shows GC chromatogram of the DBD decomposition product in Tn399, (B) shows the mass spectrum of the peak 3 in (A). It is a graph which shows GC / MS analysis of the BPH degradation product in Tn399, (A) is GC chromatogram of the BPH degradation product of Tn399, (B) is a mass spectrum of the peak 4 in (A), (C) is , (A) shows the mass spectrum of peak 5 respectively. It is a graph which shows GC / MS analysis of the 1,2-NT degradation product in Tn399, (A) is GC chromatogram of the 1,2-NT degradation product of Tn399, (B) is the peak 6 in (A). The mass spectrum of is shown. It is a graph which shows GC / MS analysis of the 2,1-NT degradation product in Tn399, (A) is GC chromatogram of the 2,1-NT degradation product of Tn399, (B) is the peak 7 in (A). The mass spectrum of is shown. It is a graph which shows GC / MS analysis of the PHE degradation product in Tn399, (A) shows GC chromatogram of the PHE degradation product of Tn399, (B) shows the mass spectrum of the peak 8 in (A). It is a graph which shows GC / MS analysis of the ANT degradation product in Tn399, (A) shows GC chromatogram of the ANT degradation product of Tn399, (B) shows the mass spectrum of the peaks 9 and 10 in (A). It is a graph to show.

Claims (8)

  A method for producing an aromatic monohydroxy product, comprising contacting an aromatic compound with a microorganism in which the enzyme activity of a polycyclic aromatic compound-degrading enzyme (bphB) is suppressed or the cell contents of the microorganism.   The aromatic monosaccharide according to claim 1, wherein the suppression of the enzyme activity of bphB is performed by at least one selected from the group consisting of suppression of bphB gene expression and gene disruption and use of a bphB activity inhibitor. A method for producing a hydroxy body.   3. The method for producing an aromatic monohydroxy substance according to claim 1, wherein the microorganism is at least one selected from the group consisting of gram-positive bacteria and gram-negative bacteria.   The method for producing an aromatic monohydroxy substance according to claim 1 or 2, wherein the microorganism is at least one selected from the group consisting of the genera Sphingomonas, Pseudomonas, and Rhodococcus.   The method for producing an aromatic monohydroxy substance according to claim 1 or 2, wherein the microorganism is a strain of the genus Sphingomonas.   The method for producing an aromatic monohydroxy substance according to claim 1, wherein the aromatic compound is selected from the group consisting of a polycyclic aromatic compound and an aromatic amino acid.   The method for producing an aromatic monohydroxy substance according to claim 1, wherein the aromatic compound is an aromatic compound having 5 to 50 carbon atoms which contains or does not contain a hetero atom.   An aromatic monohydroxy product obtained by the production method according to claim 1.