KR20030068803A - Rhodococcus sp. strain DK17 with metabolic versatility to degrade a wide variety of substituted benzenes and terpenes - Google Patents

Abstract

PURPOSE: A microorganism Rhodococcus sp. strain DK17 with metabolic versatility to degrade a wide variety of substituted benzenes and terpenes is provided, thereby effectively purifying the polluted environment without causing the second environmental pollution. CONSTITUTION: A microorganism Rhodococcus sp. strain DK17(KCCM 10332) has the activity to degrade alkyl benzenes, phenols, phthalates or terpenes, wherein the alkyl benzene compound is selected from the group consisting of o-xylene, ethylbenzene, isopropylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, toluene and benzene; the phenol compound is selected from the group consisting of phenol, o-cresol, m-cresol and p-cresol; and the phthalate compound is selected from the group consisting of phthalate or terephthalate; the terpene compound is limonene. 2,4-dimethylresorcinol, 2,5-dimethylhydroquinone or 3,4-dichlorocatechol is prepared by culturing Rhodococcus sp. DK17(KCCM 10332) together with m-xylene, p-xylene or 1,2-dichlorobenzene.

Description

Substituted benzene compounds of various structures and terpene strains D.17 {Rhodococcus sp. strain DK17 with metabolic versatility to degrade a wide variety of substituted benzenes and terpenes}

The present invention relates to a novel Rhodococcus sp. Strain DK17 which can decompose toxic organic compounds, and more particularly, o -xylene, ethylbenzene, isopropylbenzene, propylbenzene, butylbenzene. , Alkylbenzene compounds including pentylbenzene, hexylbenzene, toluene and benzene, phenol compounds including phenol, o -cresol, m -cresol and p- cresol, phthalate compounds including phthalate and terephthalate or A novel Rhodococcus strain DK17 capable of decomposing terpene compounds including limonene, a method for decomposing toxic organic compounds using the microorganisms, and a wastewater, sewage or soil treatment agent containing the microorganisms.

Aromatic compounds are harmful substances that are released after use in industrial activities or are generated as by-products during industrial activities. They can have a detrimental effect on the stable natural environment, destroying ecosystems and have a devastating effect on the human body. Among these aromatic compounds, methylbenzene is widely used industrially, including the production of chemicals, drugs, paints and enamels (Fishbein, Sci. Total Environ . 1985, 43: 165-183; Patty, Toxicology, 1963). It is known as the main substance of environmental pollution (Barbieri et al., Biodegradation , 1993, 4: 71-80). In addition, the toxic organic compounds may also have a harmful effect on the microorganisms used for the purification of wastewater and sewage, which may prevent the water purification process from occurring efficiently. Therefore, techniques for decomposing or removing these substances by using physical and chemical methods have been studied, and biodegradation methods using microorganisms among them have not been concomitant to generate contaminants. to be.

On the other hand, several bacterial strains are known that can be grown using different methyl benzenes, including three xylene isomers, as carbon and energy sources, and bacteria that break down xylene usually have m -xylene and p -xylene. They are divided into bacteria that can break down and bacteria that can only break down o -xylene. However, among the bacteria, there are rare bacteria that can perform both functions. The location of methyl groups in aromatic rings is known to play an important role in screening bacteria that can grow in xylene isomers (Barbieri et al., Biodegradation , 1993, 4: 71-80; Davis et al. , Can.J. Microbiol ., 1968, 14: 1005-1009).

Over the last few decades, many researchers have tried to elucidate the metabolism of m -xylene and p -xylene at the biochemical and molecular levels, and details of the metabolic pathways are being identified (Assinder and Willams, Adv. Microbiol. Physiol , 1990, 31: 1-69; Burlage et al., Appl. Environ.Microbiol., 1989, 55: 1323-28; Davey and Gibson., J. Bacteriol ., 1974, 119: 923-929; Gibson et. al., J. Bacteriol. , 1974, 119: 930-936; Kunz and Chapman, J. Bacteriol., 1981, 146: 179-191; Worsey and Williams, J. Bacteriol ., 1975, 124: 7-13; Zylstra, Molecular environmental biology , 1994). However, the mechanism of degradation of o -xylene is not yet clear. Several microorganisms are thought to have different metabolic pathways, including Nocardia sp. Strains (Gibson and Subramanian, Microbial degradation of aromatic hydrocarbons , 1984) and Rhodococcus sp. C125 (originally Corynebacterium strain C125, renamed in van der Meer et al., 1992) is the initial aromatic dioxygenase that converts o -xylene to cis-dihydrodiol ( cis). -dihydrodiol), but there is no direct evidence of this.

In Rhodococcus strain B3, two pathways, initiated by monooxygenase, have been found to occur simultaneously. One route passes through 2-methylbenzoic acid to 3-methylcatechol. It is involved in the oxidation of methyl groups to form 2-methylbenzylalcohol which is oxidized. The other pathway is thought to be initiated by the direct oxidation of the aromatic ring to form 2,3-dimethylphenol, but the oxidation product dimethylcatechol has not yet been identified (Bickerdike et al., Microbiology , 1997, 143: 2321-29). In Pseudomonas stutzeri strain OX1, o -xylene is decomposed through two consecutive monooxidation processes of aromatic rings to form 2,3-dimethylphenol and 3,4-dimethylphenol simultaneously. Converted to 3,4-dimethylcatechol and 4,5-dimethylcatechol, respectively (Baggi et al., Appl. Environ. Microbiol ., 1987, 64: 2473-78; Bertoni et al., Appl. Environ.Microbiol . , 1996, 62: 3704-3711).

The genus Rhodococcus is a group of bacteria that exhibits a wide range of metabolic activity, including degradation of various aromatic hydrocarbons (Warhurst and Fewson, Crit. Rev. Biotechnol ., 1994, 14: 29-73). It is known to have the ability to grow in phenyl, but little is known about the metabolism of o -xylene by the genus Rhodococcus (Vanderberg et al., Appl. Microbiol. Biotechnol ., 2000, 53: 447-452 Warhurst and Fewson, Crit. Rev. Biotechnol ., 1994, 14: 29-73; Masai et al., Appl.Environ.Microbiol ., 1995, 61: 2079-2085; Wang et al., Gene, 1995, 164 : 117-122; Asturias and Timmis, J. Bacteriol ., 1993, 175: 4631-4640).

Accordingly, the present inventors isolate a novel Rhodococcus strain capable of growing using a toxic organic compound such as an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound as a carbon source and an energy source, and using the strain, the toxic organic compound The present invention has been completed by inventing a method of decomposing

An object of the present invention is a novel Rhodococcus strain DK17 having a decomposing activity of an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound, a method for decomposing toxic organic compounds using the microorganism, and a wastewater containing the microorganism. To provide sewage or soil treatment.

1A shows the total ion chromatogram of metabolites generated during 24 hours incubation of mutant strain DK180 with o -xylene,

Figure 1b shows the mass spectrum and cleavage pattern of the metabolite at 19 minutes in the chromatogram of Figure 1a,

Figure 2a shows the HPLC elution profile of the metabolite produced when mutant strain DK180 is incubated with toluene for 24 hours,

Figure 2b shows the mass spectrum of the metabolite appearing at 18.7 minutes in Figure 2a,

Figure 2c shows the mass spectrum of the metabolite appearing at 19.9 minutes in Figure 2a,

FIG. 3 shows NMR that 2,4-dimethyllysono was produced when Rhodococcus strain DK17 was incubated with m -xylene.

FIG. 4 shows NMR that 2,5-dimethylhydroquinone was produced when Rhodococcus strain DK17 was incubated with p -xylene.

FIG. 5 shows NMR that 3,4-dichlorocatechol was produced when Rhodococcus strain DK17 was incubated with 1,2-dichlorobenzene.

Figure 6 is a photograph showing the pulsed field gel electrophoresis (PFGE) profile of the genome DNA prepared from Rhodococcus strains DK17, DK176 and DK180.

Lane 1: λ ladder, lane 2: Rhodococcus strain DK17,

Lane 3: Rhodococcus strain DK176, Lane 3: Rhodococcus strain DK180

In order to achieve the above object, the present invention provides a novel Rhodococcus sp. Strain DK17 having a decomposing activity of an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound.

The present invention also provides a method for decomposing an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound using the Rhodococcus strain DK17.

In addition, the present invention provides a wastewater, sewage or soil treatment containing the Rhodococcus strain DK17.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Rhodococcus strain DK17 having a decomposing activity of an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound.

The microbial properties of the Rhodococcus strain DK17, a novel microorganism provided by the present invention, based on the morphological, physiological and molecular biological characteristics are as follows.

Rhodococcus strain DK17 of the present invention is a Gram-positive aerobic bacterium and shows various cell types depending on growth substrate. For example, Rhodococcus strain DK17 has a short rod shape during growth in glucose and branching rather than mycelium during growth in o -xylene. As a growth titration temperature of the said strain, it is preferable that it is 20-37 degreeC, It is more preferable that it is 30 degreeC, It is preferable that pH is 6.0-8.0. In enzyme activity, it appears as oxidase-negative and catalase-positive bacteria. In addition, the strain contains an oxygenase that oxidizes indole to indigo.

16S rRNA gene of Rhodococcus sp DK17 of the present invention is a nucleotide sequence described in SEQ ID NO: 1 has a (GenBank accession number AF468521). Comparing the sequences using GenBank or Ribosome Database Project Database, the DK17 16S rRNA gene sequence shows very high homology with several other Rhodococcus species, and Rhodococcus opacus ( R. opacus ) and 96% homology.

Rhodococcus strain DK17 of the present invention is not only different in physiological characteristics from standard strains of Rhodococcus, but also has an excellent ability to decompose aromatic compounds, especially o -xylene, including ethylbenzene, isopropylbenzene, propylbenzene, Alkylbenzene compounds including butylbenzene, pentylbenzene, hexylbenzene, toluene and benzene, phenols including phenol, o -cresol, m -cresol and p -cresol, phthalates including phthalate and terephthalate The compound or terpene compound containing limonene is decomposed. In the present invention, the organic compound decomposed by Rhodococcus strain DK17 is not necessarily limited to the above.

The inventors of the novel strain, Rhodococcus sp. Strain DK17, Rhodococcus sp. Strain DK17 having a degrading activity of the above alkylbenzene compounds, phenol compounds, phthalate compounds or terpene compounds, dated November 20, 2001 It was deposited (accession number: KCCM 10332).

The present invention also provides a mutant strain of Rhodococcus strain DK17, Rhodococcus strain DK176 and Rhodococcus strain DK180.

The Rhodococcus strain DK176 and Rhodococcus strain DK180 were prepared by modified Carlton method (UV light irradiation) (Carlton and Brown, Gene mutation , 1981). Rhodococcus After inducing UV mutations using sp DK17, o-xylene and o of the selected mutants with no ability to grow strain-indole (indole) when under xylene presence grow on glucose as Indigo (indigo) A strain that lost the ability to convert and could grow in phenol but could not grow in ethylbenzene, isopropylbenzene, toluene or benzene was named "Rhodococcus strain DK176" (see Table 2 ).

The second class of UV mutants could still grow in benzene and phenol, but the strains that could not grow in alkylbenzenes were named “Rhodococcus strain DK180” (see Table 2 ).

The present invention also provides a method for decomposing an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound using the Rhodococcus strain DK17.

The degradation method of the present invention is achieved by culturing Rhodococcus strain DK17 of the present invention where the toxic organic compound is present.

The optimum conditions for effectively decomposing such compounds using Rhodococcus strain DK17 of the present invention is preferably 20 to 37 ℃, more preferably 30 ℃, pH is preferably 6.0 to 8.0. Do.

Compounds that can be decomposed by the strain of the present invention include o -xylene, ethylbenzene, isopropylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, toluene and benzene alkylbenzene compounds composed of benzene, phenol, o Phenolic compounds consisting of -cresol, m -cresol and p -cresol, phthalate compounds consisting of phthalate and terephthalate and terpene compounds such as limonene, and the like, in particular for o -xylene, ethylbenzene and terephthalate Excellent decomposition activity

In addition, the present invention by incubating the Rhodococcus strain DK17 with m -xylene, p -xylene or 1,2-dichlorobenzene to 2,4-dimethylresorcinol (2,4-dimethylresorcinol), 2, Provided are methods for preparing 5-dimethylhydroquinone (2,5-dimethylhydroquinone) or 3,4-dichlorocatechol.

When the Rhodococcus strain DK17 of the present invention is cultured in the presence of m -xylene or p -xylene, the compound is oxidized by the Rhodococcus strain DK17, respectively, to form 2,4-dimethylrisosocin or 2,5-dimethyl. Hydroquinone is produced (Scheme 1, Scheme 2; see FIG. 3, FIG. 4 ). In addition, culturing Rhodococcus strain DK17 in the presence of 1,2-dichlorobenzene causes the compound to be oxidized to produce 3,4-dichlorocatechol (Scheme 3; see FIG. 5 ).

In addition, the present invention is cultured Rhodococcus strain DK180 with o -xylene or toluene to prepare 3,4-dimethylcatechol (3,4-dimethylcatechol) or 3-methylcatechol and 4-methylcatechol Provide a method.

When cultured in the presence of o-xylene - - the Rhodococcus sp DK180 of the present invention, o-xylene is oxidized 3,4-dimethyl catechol is produced (see Scheme 4, Fig. 1). In addition, culturing Rhodococcus strain DK180 in the presence of toluene oxidizes the compound to produce 3-methylcatechol or 4-methylcatechol (see Scheme 5, Figure 2 ).

In addition, the present invention provides a wastewater, sewage or soil treatment containing the Rhodococcus strain DK17.

The treating agent of the present invention can be used to purify wastewater, sewage or soil contaminated by alkylbenzene compounds, phenol compounds, phthalate compounds or terpene compounds, and includes the microorganisms of the present invention and pharmaceutically acceptable additives. The treatment agent including the microorganism and the additive of the present invention may be directly smeared or sprayed with the culture medium of the microorganism, or the method of spraying the culture medium of the microorganism by adsorbing diatomaceous earth, perlite, or the like may be used. The treatment agent includes a novel strain of Rhodococcus strain DK17 of the present invention as an active ingredient, which may be supplied as an additive but may be stored separately for long-term storage and mixed before use. To this end, the microorganisms to be used in the treatment agent are stored at -80 ° C, including glycerol components, or suspended in sterile 10% skim milk to be freeze-dried for long term stability. Other additives included in the treatment agent may include excipients, stabilizers, and additives generally known in the biological treatment agent manufacturing field using microorganisms, or may use an adsorbent such as diatomaceous earth, perlite, loess, and the like.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> o Isolation of Strains with Degradation Activity of Xylene

The present inventors separated the growing strain using o -xylene as a substrate as the sole carbon source and energy source to isolate strains having a decomposition activity of aromatic compounds. Specifically, o -xylene decomposed strains were separated from soil contaminated with oil of Yeocheon (Korea) Industrial Complex using an enrichment method. To this end, 5 g of the soil was first added to a 250 ml flask containing 50 ml of MSB (mineral salts basal) medium (Stainer et al., J. Gen. Microbiol ., 1966, 43: 159-271). The only carbon and energy source provided o -xylene in the vapor state of the glass bulb. The flask was shaken at 200 rpm and incubated for 48 hours at 30 ° C., and 5 ml of the culture was transferred to 50 ml of fresh MSB medium for 2 days. Cultures incubated for 2 days were serially diluted and plated on MSB agar plates. The plates were incubated at 30 ° C. in the presence of o -xylene provided in steam in glass vials with cotton plugs, and the fastest growing o -xylene degrading microorganisms were selected.

As a result, the present inventors selected strains which grew rapidly using o -xylene as a substrate as the only carbon and energy source, and named the strain "DK17".

DK17 is a gram-positive aerobic bacterium and exhibited oxidase-negative and catalase-positive in enzyme activity. The growth titration temperature was 30 ° C., and the growth titration pH was 6.0 to 8.0. In addition, DK17 showed various cell types depending on growth substrate. For example, DK17 showed a short rod during growth in glucose and branching rather than mycelium during growth in o -xylene.

The present inventors amplified the 16S rRNA gene of DK17, and analyzed the nucleotide sequence of the PCR product in order to identify the above-selected DK17.

As a result, the 16S rRNA gene of DK17 was found to have the nucleotide sequence set forth in SEQ ID NO: 1 .

The inventors have identified the base sequence of the 16S rRNA gene identified above by using GenBank (Altschul et al., J. Mol. Biol ., 1990, 215: 403-410) or a ribosomal database project database. In the analysis, the DK17 16S rRNA gene sequence showed very high homology with several other Rhodococcus species, especially 96% homology with R. opacus . From the above results, DK17 having o -xylene decomposing activity isolated above was identified as Rhodococcus.

Accordingly, the present inventors named the novel strain selected above as " Rhodococcus sp. Strain DK17" and deposited it on November 20, 2001 with the Korea Microorganism Conservation Center (Accession No .: KCCM 10332)

Example 2 Degradation Activity of Organic Compounds of Rhodococcus Strain DK17

The inventors of the present invention, in order to determine whether the strain DK17 genus Rhodococcus isolated in Example 1 has a degrading activity against other organic compounds, including o -xylene, the alkylbenzene compounds, phenol compounds, phthalate compounds or terpene compounds Degradation activity was analyzed by measuring the rate of colony formation in MSB plates as the only carbon and energy source. In the case of volatile organic compounds (alkylbenzene compounds, phenol compounds, and terpene compounds), the plates were left in a sealed container containing glass vials with cotton plugs and incubated at 30 ° C. in the presence of organic compounds provided in a vapor state. The organic compound (phthalate compound) present in the powder state was dissolved in distilled water and incubated at 30 ° C. in an MSB plate at a final concentration of 5 mM. Growth and speed were determined according to the time taken to form colonies of 1.0 mm or more in diameter.

As a result, Rhodococcus strain DK17 grew well in o -xylene and produced yellow compound around colonies, forming colonies of 1.0 mm size before 3 days ( Table 1 ). The doubling time of Rhodococcus strain DK17 in MSB liquid medium provided with o -xylene vapor was 2.4 hours. Rhodococcus strain DK17 could also be grown in ethylbenzene, isopropylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, toluene and benzene, but other xylene isomers such as m -xylene and p -xylene Was not used as a growth substrate.

Rhodococcus strain DK17 also grew well in phenolic compounds including phenol, o -cresol, m -cresol and p -cresol, and phthalate compounds including phthalates and terephthalates or terpenes including limonene Grows well at. However, Rhodococcus strain DK17 could not grow in isophthalate ( Table 1 ).

compound growth Alkylbenzenes o- xylene (o -Xylene) ++++ ^a m- xylene (m -Xylene) ^-b p- xylene (p -Xylene) - Ethylbenzene +++ Isopropylbenzene +++ Propylbenzene +++ Butylbenzene +++ Pentylbenzene ++ Hexylbenzene + Toluene ++ Benzene + Phenolics Phenolic o - cresol (o -Cresol) +++ m - cresol (m -Cresol) ++ p - cresol (p -Cresol) +++ Phthalates Phthalate +++ Isophthalate - Terephthalate ++++ Terpenes Limonene +

a: 4, 3, 2 and 1 + marks indicate forming colonies of 1.0 mm diameter in 3, 5, 7 and 9 days, respectively.

b:-indicates that it cannot be used as a carbon and energy source.

<Example 3> o Metabolic Pathway Analysis of Xylene Decomposition

The inventors analyzed the growth capacity of Rhodococcus strain DK17 in a potential intermediate metabolite of o -xylene to elucidate the metabolic pathway for o -xylene degradation. Rhodococcus strain DK17 of the present invention is a hydroxylated derivative produced by direct oxidation of an aromatic ring as well as 2-methylbenzyl alcohol, 2-methylbenzylaldehyde and 2-methylbenzoic acid, which are methyl group oxidized derivatives of o -xylene. It did not grow in 2,3-dimethylphenol and 3,4-dimethylphenol. Previously, Rhodococcus strain B3 failed to grow in 2-methylbenzyl alcohol, 2-methylbenzylaldehyde, and 3,4-dimethylphenol due to its toxicity to cells caused by the above metabolites, which caused the growth of the strain in glucose. It has been reported that this was completely inhibited by this compound (Bickerdike et al., Microbiology, 1997, 143: 2321-2329). A similar inhibitory effect was observed when Rhodococcus strain DK17 was grown in glucose when 2-methylbenzylaldehyde, 2,3-dimethylphenol or 3,4-dimethylphenol was present at a concentration of 0.1%, but 2-methylbenzyl alcohol Or, although there is no inhibitory effect by 2-methylbenzoic acid, the two substances were not used as growth substrates.

From the above results, it was confirmed that Rhodococcus strain DK17 of the present invention cannot use 2-methylbenzyl alcohol or 2-methylbenzoic acid as a growth substrate.

Example 4 Preparation of Mutant Strains o Metabolic Pathway Analysis of Xylene Decomposition

We have prepared UV mutants for Rhodococcus strain DK17 to further analyze the metabolic pathways of Rhodococcus strain DK17 for o -xylene degradation. Specifically, the generation of UV mutants was performed by slightly modifying Carlton's method (Carlton and Brown, Gene mutation , 1981, p224-225). The cultured cells were centrifuged, washed twice with 0.1 M MgSO ₄ and then suspended again with the same solution. After serial dilution with 0.1 M MgSO ₄ , each cell suspension was transferred to sterile glass petri plates and exposed to 254 nm UV for 90 seconds on 22 cm of open petri plates. Treated cell suspensions were plated on MSB agar plates containing 20 mM glucose (hereinafter referred to as "master plates") and incubated in the dark at 30 ° C. Using a toothpick, transferred to the colonies formed on the MSB to the master plate to plate, this o-examined whether the growth using a xylene-o-xylene is the sole carbon and energy source, while cultured in a saturated air.

Converting the indole (indole) when grown in glucose under xylene vapor present in the indigo (indigo) - After a derive the UV mutation, o - 49 strains among the selected 54 mutant strain no ability grown in xylene o Loss of capacity, indicating impaired early oxygenase (Ensley et al., Science , 1983, 222: 167-169; Eaton and Timmis, J. Bacteriol ., 1986, 168: 123-131). Of these, the DK176 mutant strain could still grow in phenol, but not in ethylbenzene, isopropylbenzene, toluene or benzene ( Table 2 ). The results indicate that the same oxygenase is involved in the early stages in the metabolism of ethylbenzene, isopropylbenzene, benzene, toluene and o -xylene by Rhodococcus strain DK17.

The second class of DK180 mutant strains of these UV mutants could still grow in benzene and phenol, but not in alkylbenzene ( Table 2 ). While methylbenzene is converted to methylcatechol, aerobic decomposition of benzene proceeds via catechol. Thus, failure of Rhodococcus strain DK180 to grow in o -xylene or toluene indicates that the enzyme in the step of decomposing methylcatechol in the strain is destroyed.

The results indicate that the normal oxidase process works during the initial oxidation of alkylbenzenes by Rhodococcus strain DK17 but has at least two different pathways for the decomposition of (methyl) catechol.

compound DK17 DK176 DK180 o -xylene ++++ ^a _ - m -xylene ^-b - - p -xylene - - - Ethylbenzene +++ - - Isopropylbenzene +++ - - toluene ++ - - benzene + - + phenol +++ +++ +++

a: 4, 3, 2 and 1 + marks indicate forming colonies of 1.0 mm diameter in 3, 5, 7 and 9 days, respectively.

b:-indicates that it cannot be used as a carbon and energy source.

Example 5 Induction of meta- and ortho-cutting deoxygenase in DK17 and DK180 strains

Methylcatechol is known to be metabolized more efficiently by the meta-cleavage pathway than the ortho-cleavage route because of alkyl substitution (Harayama et al., J. Bacteriol. , 1987, 169: 558-564). Thus, we analyzed what type of ring-cut dioxygenase was physiologically induced during normal DK17 strain and mutant strain DK180 in the presence of o -xylene, toluene or benzene.

Specifically, DK17 and DK180 cells cultured in 20 mM glucose were harvested and resuspended in 200 ml fresh MSB medium. To induce the ring-cutting pathway, o -xylene, toluene or benzene was added directly to a final concentration of 0.1% and it was incubated at 30 ° C. for 12 hours. The induced cells were harvested and washed with about half the volume of 1 × PBS (phosphate-buffered saline; 140 mM NaCl, 2.7 mM KCl, 10 mM NaHPO ₄ , 1.8 mM KH ₂ PO ₄ [pH 7.4]), and 5 ml of Suspended in 50 mM MOPS (1 mM ascorbic acid, 10% acetone, 10% glycerol and 100 uM FeSO ₄ , pH 7.8) and triturated by ultrasound. Unmilled cells and cell debris were removed by centrifugation at 10,000 × g for 30 minutes and the remaining supernatant was used as enzyme solution.

Catechol 2,3-dioxygenase (C23O) activity was analyzed by measuring the increase in absorbance at the maximum absorption wavelength of each meta-cut product formed from the following substrate; Catechol, λ _max = 375 nm and ε = 33,400 cm ⁻¹ M ⁻¹ ; 3-methylcatechol, λ _max = 388 nm and ε = 13,800 cm ⁻¹ M ⁻¹ ; 4-methylcatechol, λ _max = 382 nm and ε = 28,100 cm ⁻¹ M ⁻¹ (Bayly et al., Biochem. J., 1966, 101: 293-301). The reaction mixture contains 100 mM phosphate buffer (pH 7.4) and a suitable substrate with a final concentration of 0.4 mM. The activity of catechol 1,2-deoxygenase (C12O) was analyzed by measuring the increase in absorbance at the maximum absorption wavelength of each ortho-cutting product formed from the following substrates; For cis, cis-muconate 260 nm: ε = 16,800 cm ^-1 M ^-1 ; For 2-methyl-cis, cis-muconate 260 nm: ε = 18,000 cm ^-1 M ^-1 ; For 3-methyl-cis, cis-muconate 255 nm: ε = 14,300 cm ^-1 M ^-1 (Dorn and Knackmuss, Biochem. J., 1978, 85-94). Protein content was determined using bovine serum albumin as the standard protein according to the Bradford method (Bradford, Anal. Biochem., 1976, 72: 248-254). One unit of enzyme activity was defined as 1 umol product / min / mg (protein).

As a result, a large amount of meta-cutting deoxygenase (C23O) activity was detected in the cells when the normal Rhodococcus strain DK17 was grown in o -xylene, toluene or benzene, but under the same conditions, No cleavage deoxygenase activity was detected ( Table 3 ). Meta-cleaved deoxygenase detected had the highest activity for 3-methylcatechol and high activity for 4-methylcatechol (30.5% level of 3-methylcatechol), but not for catechol. Showed very low activity (a level of 1.9% of 3-methylcatechol). It is clear that the same meta-cleaving enzyme is induced in the presence of these three substrates because of the same ratio of activity in benzene, toluene and o -xylene.

Strain Inductance Enzyme activity (U / mg of protein) ^a Catechol 3-methylcatechol 4-methylcatechol DK17 Glucose ND ^b ND ND o -xylene 32.0 ± 1.8 1,666.3 ± 71.1 507.9 ± 11.1 toluene 15.8 ± 2.3 818.6 ± 78.9 253.9 ± 29.9 benzene 6.8 ± 2.3 226.3 ± 27.3 60.1 ± 10.7 DK180 o -xylene ND ND ND toluene ND ND ND benzene ND ND ND

a: displays average enzyme activity in at least three independent experiments,

b: not detected

However, ortho-cutting deoxygenase (C12O) was induced only when Rhodococcus strain DK17 grew in the presence of benzene ( Table 4 ), and also mutant strain DK180 without normal and meta-cutting deoxygenase activity. To the same level. This would lead to the growth of Rhodococcus strains DK17 and Rhodococcus strain DK180 in benzene with low levels of meta-cutting deoxygenase against catechol and reduced levels of meta-cutting deoxygenase induction in the presence of benzene. When catechol, the same amount of metabolic flux occurs. This is not a new pathway of metabolism through other downstream pathways in mutant strains.

The enzyme analysis data demonstrated that the failure of Rhodococcus strain DK180 to grow in o -xylene was a result of mutation of the gene encoding meta-cleaved deoxygenase.

Strain Inductance Enzyme activity (U / mg of protein) ^a Catechol 3-methylcatechol 4-methylcatechol DK17 Glucose ND ^b ND ND o -xylene ND ND ND toluene ND ND ND benzene 577.0 ± 5.6 55.1 ± 7.2 156.7 ± 13.5 DK180 benzene 480.3 ± 18.9 63.6 ± 14.7 185.5 ± 24.8

a: displays average enzyme activity in at least three independent experiments,

b: not detected

Example 6 Preparation of Compound Using DK17 and DK180 Strains

<6-1> Preparation of Compound Using Rhodococcus strain DK180

We incubated Rhodococcus strain DK180 with o -xylene or toluene to prepare 3,4-dimethylcatechol or 3-methylcatechol and 4-methylcatechol.

Specifically, mutant strain DK180 produced by UV was incubated overnight at 30 ° C. in MSB medium containing 20 mM glucose. 10 ml of the culture was transferred to 400 ml fresh medium containing 5 mM glucose and incubated for 24 hours under the same conditions in the presence of o -xylene or toluene provided in the vapor state. Cells were removed by centrifugation at 10,000 × g for 30 minutes and the supernatant was extracted twice with the same volume of ethyl acetate. After concentration by rotary evaporator, it was dissolved in a small amount of methanol and analyzed by HPLC (high pressure liquid chromatography). HPLC analysis was performed with an HPLC instrument (Hewlett-Packard model 1100 HPLC) mounted on a 5 μm ZORBAX column (4.6 × 250 mm). The mobile phase was a 45 minute gradient of methanol-water (5: 95-95: 5) at a flow rate of 1.0 ml / min. Gas chromatography-mass spectrometry (GC-MS) analysis of metabolites was performed by mass spectrometry (Hewlett-Packard 5973, linked to a 6890 gas chromatogram suitable for fused silica capillary columns (HP-5, 0.25X30 mm, 0.25 μm film thickness). electron impact ionization (70 eV). GC was performed under the following conditions: 1 mL He / min, on-column injection mode; Oven temperature, 60 ° C. for 2 minutes; The temperature change was also 5 ° C./min up to 220 ° C., followed by 220 ° C. Prior to injection, the samples were subjected to N -methyl- N -trimethylsilyltrifluoroacetamide, methane, which resulted in pyridine to produce trimethylsilyl ether (TMSi), methaneboronate (MB) or phenanthreneboronate (PB) derivatives, respectively. Boronic acid or 9-phenanthreneboronic acid was treated at 80 ° C. for 30 minutes. Acetylation was performed to stabilize the o -xylene metabolite prior to NMR analysis. The dried product was dissolved in acetic anhydride (1 mL) and pyridine (2 mL) and incubated overnight at room temperature. The product was then adjusted to pH 7.0 with HCl solution and extracted with 30 mL n -hexane. After removal of n -hexane, the acetate of the metabolite was collected and applied to normal phase HPLC (6 × 150 mm, Senshu Pak Aquasil SS-532N) at a flow rate of 1 mL / min with a mixture of n -hexane-isopropanol as the elution solvent. (0-5 min; 100% n -hexane, 5-25 min; gradient from n -hexane to 5% iso-propanol; then n -hexane to 5% iso-propanol). The sample was dissolved in DCDl ₃ and analyzed by 300 MHz ¹ H NMR (Varian Gemini 2000) using tetramethylsilane as an internal standard solvent.

As a result, the metabolites m / z m / z 193 ( M + -OTMSi) generated by the fragmentation of molecular ions and molecular ions at 282 and has a major ion, which is originally a metabolite di hydroxyl rate of o - Implies xylene. To determine if a dihydroxyl group is present in the immediate position, the metabolite was derived with methaneboronic acid or 9-phenansrenboronic acid which form a boronate derivative only when it acts with a dihydroxyl group located nearby. Two hydroxyl groups are bound to o -xylene, and GC-MS confirmed the location of the two hydroxyl groups. Methaneboronic acid and 9-phenan styrene boronic acid must be present in two adjacent hydroxyl groups to form a methane boronate and phenanthrene boronate derivative. GC-MS data confirmed that the two hydroxyl groups were adjacent ( FIG. 1A ).

Two hydroxyl groups are o - there is coupled adjacent to the xylene, then o - is examined by NMR analysis exists in any position and two methyl groups in the xylene. The expected molecular ions for the methaneboronate and phenanthrenboronate derivatives of the metabolite were measured continuously at m / z 162 and m / z 324, respectively. Thus, the metabolite at 19.0 min is 1,2-dihydroxy-3,4-dimethyl-benzene (3,4-dimethylcatechol) or 1,2-dihydroxy-4,5-dimethyl-benzene ( 4,5-dimethylcatechol). Purification of the main metabolite for further NMR analysis was not possible because the extracted metabolite was very unstable at room temperature. To solve this problem metabolites were stabilized by acetylation and purified by normal phase HPLC and analyzed by 300 MHz ¹ H-NMR. As summarized in Table 5 , the acetate derivatives of the major metabolites gave a singlet at δ2.27 for 6H (6 protons), which assigned two equivalent methyls in diacetyl, o -xylene Two methyls derived from were detected at δ2.08 (s, 3H) and δ2.32 (s, 3H). The two mutually bound aromatic protons were absorbed at δ7.05 (d, J = 8.3 Hz) and δ6.92 (d, J = 8.3 Hz), which attached the aromatic protons to C-5 and C-6 It means that there is. Based on GC-MS and NMR data, the main metabolite was found to be 3,4-dimethylcatechol (Scheme 4; FIG. 1B ).

<Scheme 4>

Proton (s) ppm 2CH ₃ -1,2-OAc 2.27 (s) CH ₃ -3 2.32 (s) CH ₃ -4 2.08 (s) H-5 6.92 (d, J = 8.3 Hz) H-6 7.05 (d, J = 8.3 Hz)

As shown in the enzyme assay data (see Table 3 ), toluene is degraded via meta-cutting pathways such as o -xylene. The HPLC elution profile of the ethyl acetate-extracted metabolite of toluene formed by DK180 is shown in FIG. 2A . Two major peaks eluted at 18.7 min and 19.9 min. GC-MS analysis of each peak showed the same weight spectra and GC retention times as commercial compounds, so the intermediate metabolites detected at 18.7 min and 19.9 min were 3-methylcatechol and 4- Identified as methylcatechol (Scheme 5; FIGS. 2B and 2C) .

Scheme 5

<6-2> Preparation of Compound Using Rhodococcus Strain DK17

The present inventors incubated Rhodococcus strain DK17 with m -xylene, p -xylene or 1,2-dichlorobenzene in the same manner as in Example <6-1>, to provide 2,4-dimethylrisosinol , 2,5-dimethylhydroquinone or 3,4-dichlorocatechol was prepared. The compound prepared above was prepared in the same manner as in Example <6-1> using 2,4-dimethylisocinol (Scheme 1; FIG. 3 ), 2,5-dimethylhydroquinone through GC-MS analysis and NMR analysis . (Scheme 2; FIG. 4 ) or 3,4-dichlorocatechol (Scheme 3; FIG. 5 ).

<Scheme 1>

<Scheme 2>

<Scheme 3>

Example 7 PFGE Separation of Genome DNA from DK17, DK176, and DK180

Of the 54 mutant strains that do not grow in o -xylene, interestingly, 49 mutant strains, including DK176, show the same phenotype (loss of early oxygenase). In addition, revertants could not be obtained from mutant strains of this classification. This suggests that the early oxygenase gene of DK17 is present in the plasmid and the plasmid is lost by UV. The present inventors performed PFGE (pulsed-field gel electrophoresis) analysis of genome DNA to separate large plasmids from different strains.

Specifically, agarose plugs containing genome DNA were prepared by slightly modifying the Saeki method (Saeki et al., Microbiology, 1999, 145: 1721-1730). Harvest bacterial culture and resuspend in EET (0.1 M EDTA, 10 mM ethylene glycol-bis ( β -aminoethyl ether) -N, N, N'N'- tetraacetic acid, 10 mM Tris / HCl, pH 8.0) And mixed with 2.0% (w / v) agarose (low melting agrose). Plug into TE buffer (50 mM Tris, 20 mM EDTA, pH 8.0) containing lysozyme (2 mg / ml), protease K (2 mg / ml) and sodium N -leroylsacocine (0.5 mg / ml) After incubation for 48 hours at 30 ℃ was incubated for 12 hours at 55 ℃ using EET buffer containing 1% (w / v) SDS. The plug was washed three times in TE buffer and stored at 4 ° C. in the same buffer. PFGE was performed using the Bio-Rad Laboratories CHEF DR III system. Gels (1.0% agarose in 0.5X TBE buffer [1X TBE was 89 mM tris borate, 2.5 mM EDTA, pH 8.0]) were electrophoresed at 14 V at 6 V / cm. Pulse duration increased from 15 to 60 seconds for 16 hours of electrophoresis.

As a result, as shown in Figure 6 Rhodococcus strain DK17 of the present invention contained at least two large plasmids, pDK1 (about 380 Kb) and pDK2 (about 330 Kb). In addition, pDK2 was lost in the mutant strain DK176. The results indicate that structural genes for early oxygenase and meta-ring cleavage oxygenase are present in pDK2.

As described above, the Rhodococcus strain DK17 of the present invention is an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound which is leaked into the natural environment during industrial activities and thus has a harmful effect on humans and organisms and further causes environmental pollution. Since the microorganism is applied to waste water, sewage or soil contaminated by the compound, it can be usefully used to efficiently clean the environment without causing secondary environmental pollution through biodegradation. Major enzymes and genes involved in this biodegradation process (eg aromatic oxygenase) can be applied to the development of new bioconversion systems for the production of high value-added compounds.

Claims (9)

Rhodococcus sp. Strain DK17 (Accession No .: KCCM 10332) having a decomposing activity of an alkylbenzene compound, a phenol compound, a phthalate compound or a terpene compound. The method of claim 1, wherein the alkylbenzene compounds are selected from the group consisting of o -xylene, ethylbenzene, isopropylbenzene, propylbenzene, butylbenzene, pentylbenzene, hexylbenzene, toluene and benzene Caucasus strain DK17. The genus Rhodococcus strain DK17 according to claim 1, wherein the phenolic compound is selected from the group consisting of phenol, o -cresol, m -cresol and p -cresol. The genus Rhodococcus strain DK17 according to claim 1, wherein the phthalate compound is phthalate or terephthalate. The genus Rhodococcus strain DK17 according to claim 1, wherein the terpene compound is limonene. Rhodococcus strain DK17 of claim 1 was incubated with m -xylene, p -xylene or 1,2-dichlorobenzene to obtain 2,4-dimethylresorcinol, 2,5-dimethyl Method for preparing hydroquinone (2,5-dimethylhydroquinone) or 3,4-dichlorocatechol (3,4-dichlorocatechol). The Rhodococcus strain DK180 of claim 1, wherein the Rhodococcus strain DK17 is mutated by UV and has a degradation activity of benzene or toluene. A method for preparing 3,4-dimethylcatechol or 3-methylcatechol and 4-methylcatechol by incubating Rhodococcus strain DK180 of claim 7 with o -xylene or toluene. A wastewater, sewage or soil treatment comprising the Rhodococcus strain DK17 of claim 1.