KR100563207B1 - Method for Biological Degradation of Alkylphenol Causing Environmental Pollution - Google Patents

Abstract

The present invention relates to a method for decomposing alkylphenols used as a surfactant and polluting the environment, wherein the single or mixed strains of large tooth mushrooms or pore mushrooms are contacted with alkylphenols to decompose the alkylphenols. Provide a biological degradation method.

Description

Biological Degradation Method of Alkylphenols That Cause Environmental Pollution {Method for Biological Degradation of Alkylphenol Causing Environmental Pollution}             

1 is a result of measuring the resistance of pore mushrooms and large tooth mushrooms to octylphenol

2 is a measurement result of the resolution of the culture period of octyl phenol by the big tooth mushroom and pore mushroom

Figure 3 is the result of measuring the MnP induction effect of pore mushroom and big tooth mushroom by the addition of octyl phenol

Figure 4 is the result of measuring the laccase induced effects of pore mushrooms and big tooth mushrooms by the addition of octylphenol

5 is a result of measuring the pH change of the pore mushroom and big tooth mushroom medium by the addition of octylphenol

Figure 6 is the result of measuring the cell proliferation of octyl phenol products degraded by pore mushrooms

7 is a result of measuring the cell proliferation of octyl phenol products decomposed by large mushrooms

8 is a result of measuring the effect of octylphenol products degraded by pore mushrooms on pS2 gene expression

Figure 9 is the result of measuring the effect of ps2 gene expression of octylphenol products degraded by big tooth mushroom

The present invention relates to a decomposition method of alkylphenols used as a surfactant and pollutes the environment, and more particularly to alkylphenols, especially octylphenol (4-t-octylphenol), used as surfactants to pollute water and soil. The present invention relates to a biological decomposition method using specific wood lysing bacteria having excellent resolution.

Xenoestrogen, a substance that enters the body from the environment and acts similar to the female hormone estrogen, is called xenoestrogen. In general, most of the endocrine obstacles are defined as such substances, and among the environmental pollutants, alkylphenols are representative. Although alkylphenols do not have a steroid structure similar to estrogens, they are known to cause various physiological effects similar to estrogens by binding to the estrogen response element (ERE) of the estrogen receptor in the body. Among these alkylphenols, 4-pentylphenol, 4-octylphenol, and nonylphenol isomers are mainly used for the use of antioxidants, stabilizers, and nonionic surfactants in synthetic resins such as plastics, and thus environmental factors caused by water pollution and eating habits. In most cases, it flows into the body.

Recently, researches to decompose such environmental pollutants using biotechnological methods have been intensively conducted, and in particular, microorganisms of biodegradable substances related to alkylphenols such as bisphenol A, nonylphenol, and phthalates using bacteria and plastic plasticizers are used. Decomposition studies have been the main subjects, and after the confirmation of the degradation of penanthracene using white fungi Phanerochaete chrysosporium , studies to decompose difficult-decomposable substances related to endocrine disruptors using white fungi have continued. Is being performed. I related the recent White rot fungi decompose substances decompose study tested Rhine-mail Bercy Color, Rhine-mail Tess Bill Rosa, Lotus Osteria flu research has been conducted on a variety of white rot fungi such as ahruseu. In addition, in Korea, studies have been continuously made to remove hardly degradable environmental pollutants which are various types of phenol chlorides using native white fungi.

In the case of the decomposition of hardly decomposable substances using white fungus, it is very important in the difference that it has a system that can proceed the decomposition regardless of the types of hardly decomposable substances, unlike the method using microorganisms such as bacteria. In the case of bacteria, although the resolution is related to a specific hardly decomposable substance, the resolution decreases with respect to other kinds of substances. However, in white rot bacteria, an extracellular enzyme is used to maintain the degradation system, which leads to continuous degradation. Is done. Major enzymes related to this include lignin peroxidase, manganese-dependent peroxidase and manganese-independent peroxidase, and laccase, which are well known as enzymes for selectively removing lignin from the wood. It is confirmed that various enzymes such as cellulase and oxidase related to the growth environment are being operated, which is an opportunity to confirm the importance of white fungus in the biological recovery field.

However, the discovery of new strains that have the ability to reduce the estrogen properties of degradation products with excellent resolution of these substances with respect to alkylphenols that are used as surfactants to cause environmental pollution are very few.

The present invention has been made to solve the problems of the prior art, the purpose of which is used as a surfactant is excellent resolution for alkylphenols, especially octylphenol (4-t-octylphenol) that pollute the water quality and soil, In addition, the present invention provides a method for biologically decomposing alkylphenols, which is excellent in reducing estrogen properties of degradation products.

In order to achieve the above object, the present invention is used as a surfactant, and in the decomposition method of alkylphenols that pollute the environment, decomposing the large tooth mushrooms or pore mushrooms alone or mixed strains thereof by contacting them with alkylphenols. It provides a biological decomposition method of the alkylphenols, characterized in that.

The present invention preferably provides a method for biological decomposition of alkylphenols, wherein the alkylphenols are octylphenol (4-t-octylphenol).

Hereinafter, the content of the present invention will be described in more detail.

The big tooth mushroom or pore mushroom according to the present invention is used as a surfactant as a mushroom belonging to the bacterium, and is very useful in biological recovery of alkylphenols, especially alkylphenols, which pollute the environment. This property is confirmed from the results of measuring the resistance of the alkylphenol to the strain and the resolution of the alkylphenol according to the culture, and also the biological recovery from the results of verifying the estrogen properties of the degradation products resulting from degradation of the alkylphenol. It is very useful as a method.

Big tooth mushrooms and pore mushrooms each have excellent resolution of alkylphenols by treatment alone, and can reduce the estrogen properties of the decomposition products, and these effects can be further doubled when both strains are mixed. I think you can.

As a specific method for treating alkylphenols, various methods may be sought as needed, but a method of directly culturing the mycelium of the mushrooms and directly contacting the alkylphenols may be used, or a bioreactor using enzymes secreted by them. The process of making and treating to alkylphenol can be used.

In a preferred embodiment of the present invention, among the alkyl phenols, octyl phenol (4-t-octylphenol) was examined. As a result of measuring the resolution of octylphenol, the pore and big tooth mushrooms had a difference in resolution at the beginning of the culture, but there was no significant difference as the degradation progressed, and both showed more than 95% resolution.

In addition, according to the present invention, the result of measuring the effect of the degradation product of octylphenol on the proliferation of MCF-7 cells, which are female hormone sensitive and breast cancer cells, and the expression level of pS2, which is a marker gene of MCF-7 cells, was measured. The fact of being reduced has been verified.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

Example 1 Measurement of Resistance and Resolution to Octylphenol (4-t-octylphenol)

Octylphenol (4-t-octylphenol), a surfactant in the Endocrine Disruptors List, was selected as a target material. For screening of strains, screened by RBBR method among 95 species of botanical strains and 38 kinds of edible forest mushrooms, National Forest Research Institute of Korea, and then examined the titer of lignin degrading enzyme and resistance of each concentration, Basidioradulum molare Hole mushroom ( Schizopora paradoxa ) was selected.

YMPG medium containing 100, 200, 300, 400, 500 ppm of octylphenol [1 L of distilled sugar, glucose 10 g, malt extract 10 g, bactopeptone 2 g, yeast extract 2 g, asparagine 1 g, KH ₂ PO ₄ 1 g, MgSO ₄ ㆍ 1H of 7H ₂ O, 1 mg of thiamin, and 20 g of Bacto agar were inoculated, respectively, and the growth resistance of each strain was measured by measuring the growth rings of mycelia grown daily at 30 ° C. for 14 days.

After the highest mycelial growth of each strain grown in YMPG medium, 50 μl of 20 mM stock solution of octylphenol was added to a flask containing 10 ml of SSC (slow stationary culture) liquid medium and the concentration of the compound in the liquid medium was 100 μM. Adjusted to incubation at 30 ° C. 1, 3, 6, and 12 days of incubation, 20 ml of hexane was added, and then shaken vigorously for 5 minutes, and allowed to stand for 10 minutes. The hexane layer was recovered using a pipette. The aqueous layer was added with 10 ml of ethyl acetate and 10 ml of n-hexane again, re-extracted, mixed with the primary extract and filtered using a 0.45 μm microfilter, followed by a Waters symmetric column. Was quantified by HP 1100 series HPLC and detectors were measured at UV 240 and 280 nm. Analysis conditions were flow rate 0.6ml / min, solvent was CH ₃ CN: Water = 80:20 (v / v) was analyzed under isocratic conditions (isocratic).

The results of measuring the growth cycle of mycelia for 14 days by inoculating the pores of mushrooms and big tooth mushrooms in a medium containing 100, 200, 300, 400, and 500 ppm of octylphenol are shown in FIG. 1. The mycelia were completely covered with petri dishes and considered to be full growth when the length of the growth ring was 8.5 cm.

In the resistance study of octylphenol, the pore mushroom reached full mycelial growth within 14 days at the concentration of 200ppm or higher, and the big tooth mushroom showed the limited growth gradually with the increase of concentration, and the growth was excellent even at the high concentration of 500ppm.

In the investigation of the resolution of octylphenol, pore mushrooms were degraded 95% on the first day of culture, and big tooth mushrooms showed 36% resolution, but both strains were degraded more than 95% after 3 days. (Figure 2)

Example 2 Culture Conditions for Maximizing Enzyme Production

The titers of manganese peroxidase (MnP), and laccase (Laccase) were measured by the following method. MnP titers were measured using ABTS (0.08 g / L), H ₂ O ₂ (0.1 mM), MnSO ₄ (0.2 mM), and 0.2 M lactate buffer (pH 4.5) solutions, with the last addition of the crude enzyme solution. The measurement was started. For laccase titers, only ABTS (0.8 g / L) and 0.1 M sodium lactate buffer were used. The titers of MnP and laccase were calculated by applying ε414 = 36,000M ⁻¹ cm ⁻¹ of ABTS. In addition, in order to observe the titers of MnP and laccase enzymes changed by octylphenol, the titer was investigated by the above method for 6 days every day after inoculating the strain into SSC liquid medium and 12 days after the addition of the target substance. .

In order to select the proper temperature and medium for the large tooth mushroom and the pore mushroom selected by the resistance investigation, inoculated in 2% malt extract agar and YMPG medium, and incubated at 17, 25, 30, and 34 ° C, respectively, to measure mycelial growth. Was investigated. In addition, in order to measure the change in enzyme titer according to the carbon source and nitrogen source, the medium content of glucose and asparagine was incubated in SSC medium, and the titer was measured by filtration of crude enzyme solution at the time when the mycelia reached maximum growth.

The filtrate was finally concentrated to 100 μl while bubbling with nitrogen gas, derivatized with N, O-bis (trimethylsilyl) trifluoroacetamide, and then reacted at 60 ° C. for 1 hour, followed by GC / MS (Shimadzu HiCap -CBP1-M25-025 column, 25 m, 0.25 μm, 0.25 mm). GC analysis conditions He was the carrier gas flow rate 1.0ml / min, injector 270 ℃, detector 285 ℃, oven temperature was maintained for 5 minutes at 80 ℃, 8 ℃ / min to 160 ℃ after holding for 5 minutes, again 5 ℃ The analysis was performed using a program maintained for 5 minutes after rising to 280 ° C./min. Eluent from the GC column was directly connected to MS and spectra were obtained by EI mode, 70 eV ionization energy, 50-800 amu scan for 2 seconds. Analysis of the obtained product of the spectra was confirmed by comparing with the mass spectra library (Wiley Registry of Mass Spectra Data, 6th ed.) Built in the MS system.

The effect of inducing MnP and laccases on the porcelain mushroom and big tooth mushroom by the addition of octylphenol is shown in FIG. 3. The addition of octylphenol showed the MnP induction effect by increasing MnP titer after cultivation compared to before cultivation. Decreased. Lacasees showed the highest titers before addition of both strains (FIG. 4).

The pH change of the pore mushroom and giant tooth mushroom medium by the addition of octylphenol is shown in FIG. 5. The mushrooms were reduced to 3.8 before the addition of octylphenol at pH 4.8 before incubation and maintained at 3.8 to 3.6 after addition.

Both strains of the fungi and big tooth mushrooms reached the optimum growth in the initial pH 5.0, temperature of 30-34 ℃, YMPG medium, and the maximum growth in the basic medium using 10g of glucose and 1g of asparagine in the growth change according to the carbon source and nitrogen source. Reached.

Example 3 Estrogen Reduction Effect on Target Substances and Degradation Products

The medium was added with Dulbecco's modification of Eagle's medium (DMEM) and 1mM of sodium pyruvate and 7.5% NaHCO ₃ in 1 L of distilled water, and then adjusted to pH 7.4 and filtered through a 0.2 μm filter. Then 50 ml of inactivated (56 ° C., 30 min) FBS was added to prepare a final 5% FBS DMEM medium. Contamination of the prepared medium was confirmed by the presence or absence of the medium after incubation for 48 hours in a 37 ℃ incubator. To recover cultured cells, 0.25% trypsin solution and 0.2% EDTA were prepared, respectively, and PBS: 0.2% EDTA: 2.5% trypsin (8: 1: 1, v / v / v) were mixed and filtered to 0.2 μm. It was used for the experiment.

After aspirating and removing the medium of a 25 cm 3 flask containing corn (Corning # 430168), the resultant was washed with 5 to 6 ml of PBS buffer to remove residues remaining on the bottom surface. Then, 0.25% trypsin solution was incubated for 1 to 2 minutes by adding 500 µl per flask. When MCF-7 cells were separated from the bottom of the flask, 5 ml of the medium was pipetted into the flask, and the cells were dispensed into 15 ml centrifuge tubes (Falcon # 352097) and centrifuged (1000 rpm, 4 ° C., 5 minutes). The supernatant was aspirated off with a pressure reducer, fresh medium was added and pipetted to unify the cells, and the number of cells was measured. 5㎖ into a 5% FBS DMEM media in a new 25㎤ plast, 4~5 × 10 ⁴ cells / to the cells after the division so that the plast flask slowly so that by shaking the cells are uniformly dispersed in 5% CO _2, Incubated in a 37 ℃ incubator. Once every 3 to 4 days, the medium was aspirated and removed with a pressure reducer and replaced with fresh medium. Cells were passaged at 6-8 day intervals.

In order to remove the cell growth factor contained in the serum was treated as follows. Distilled water of the serum amount × 1.1mL was put into the glass centrifuge tube with a lid, and the distilled water was removed after marking exactly on the position of water surface. 5% activated carbon (serum amount x 1.1 ml) was added, distilled water was added to the indicated point, shaken up and down to disperse the activated carbon, and then centrifuged (250 rpm, 2 minutes). The same operation was repeated three more times to remove activated carbon suspended particles. Then, 0.5% dextran (serum amount x 1.1 ml) was added, distilled water was added to the indicated point, shaken up and down, and the activated carbon was dispersed and centrifuged (600 rpm, 5 minutes). After the supernatant was aspirated and removed, serum was added and the mixture was slowly stirred to prevent foaming, thereby dispersing activated carbon. This was incubated for 60 minutes in a shaking water bath at 37 ℃, centrifuged (3000rpm, 20 minutes). The supernatant was aspirated and removed with an eyedropper and then dispensed into another glass centrifuge tube, followed by centrifugation (3000 rpm, 20 minutes). The supernatant obtained by repeating this process twice was filtered through a 0.45 μm filter. Then, the filter was sterilized by a 0.2 µm filter. After dispensing about 4 ~ 5ml in a 10ml glass centrifuge tube and stored at -20 ℃ and thawed at room temperature in the experiment, 5% of activated carbon treated serum was added to the phenol red-free medium was used for the E-screen assay.

Before dispensing cells with 96-well culture crate (Falcon # 353072, Non-pyrogenic), change the medium of the cells to be used for the test, remove the medium in the flask containing the cells once on the day of the experiment, and use PBS buffer. After washing, 0.25% trypsin was incubated for 1 to 2 minutes by adding 500 µl per flask. Then, 5 ml of medium was added and pipetted to separate the cells. The medium was collected in a 15 ml conical tube, centrifuged at 4 ° C. (1000 rpm, 5 min), and the supernatant was removed with a pressure reducer. After a certain amount of medium was added according to the density of the cells, the cells were singulated by pipetting and the number of cells was measured using a homocytometer. The cell number was 5 × 10 ³ cells / well, and a predetermined amount (100 μl) of cells were dispensed into 96 well plates (Falcon # 353072, Non-pyrogenic). The 96 well plates were shaken slowly to uniformly distribute the cells and incubated for 24 hours in a 37 ° C. incubator with a constant 5% CO ₂ , and then the medium contained in the wells was removed. 90 μl of DMEM containing Charcoal-dextran activated FBS (FBS) activated with 5% charcoal-textlan, a test medium, was added to each well, and the concentration of the test substance (octylphenol) was prepared. Added. At this time, DMSO was added as a negative control, and the final concentration of DMSO per well was not more than 0.5%. Subsequently, 96 well plates to which test substances were added were incubated for 6 days (144 hours) in a 5% CO ₂ , incubator, MCF-7 cells were grown, and total RNA was isolated. It was measured by.

Cells were prepared as in the Proliferation assay and the calculated cells were divided into 6 well plates to 5 × 10 ⁴ cells per well and incubated in a 5% CO ₂ , 37 ° C. incubator for 24 hours. After 24 hours, the medium in the well was aspirated and removed, and then washed once in the well with 5 ml of PBS. 2 ml of DMEM (without phenol red and 5% charcoal-dextran-treated DMEM with phenol) was added 2 ml each. Busy. After 72 hours, the steroid-free medium in the plate was treated in the same manner as the cell division test, but two wells per group were designated. After 24 hours, the medium in the plate was aspirated and washed once with PBS, and then 1 ml of trizol solution (Gibco BRL) was added per group (2 wells), followed by sufficient pipetting. Transfer the pipetted solution to a 1.5 ml tube, add 200 µl of chloroform, shake it sufficiently, centrifuge (14,000 rpm, 10 minutes), collect the supernatant, transfer to a new tube, add isopropanol, and mix up and down 4-6 times. Then centrifuged again (14,000 rpm, 10 minutes). After checking the pellet, the supernatant was removed with a micropipette, the tube was washed with 70% ethanol (diluted with DEPC treated DW), settled by centrifuge and the remaining ethanol was removed with a micropipette. Distilled water treated with an appropriate amount of DEPC was added (about 15 µl) and pipetted to dissolve the pellets. The concentration was measured at 600 nm ultraviolet and cDNA was synthesized using M-MLV reverse transcriptase (Ambion) with about 4 μg of total RNA as a template. PCR was performed using Tag DNA polymerase as a template of the synthesized cDNA, and front primer 5'-GGCCACCATGGAGAACAAGG and rear primer 5'-CCACGAACGGTGTCGTCGAA were used to amplify the pS2 gene. Each cycle was subjected to a deformation step at 95 ° C. for the first 5 minutes, modified at 95 ° C. for 1 minute, annealing at 50 ° C. for 1 minute, polymerization for 1 minute 30 seconds at 72 ° C., and extension repeated 30 times at 72 ° C. for 15 minutes. . The housekeeping gene was used as human 1A, and the front primer 5'-GATATGGCGTTTCCCCGCATA and the back primer 5'-GGATTTTGGCGTAGGTTTGGT were used for amplification. 50% of the PCR product was loaded on agarose gel (pS2; 2% agarose, 1A, 1% agarose) and operated at 100 volts for about 30 minutes. Staining was performed using EtBr, and the images were analyzed using Gel doc 1,000. The measured pS-2 mRNA values were normalized to 1A mRNA.

In the present invention, the effect on the proliferation of MCF-7 cells, which are human breast cancer cells, was investigated using 17β-estradiol, which is known as an estrogen agonist, as a positive control. After the test material was inoculated with the first 200 ppm of octylphenol, and then cultured with pore mushrooms and big tooth mushrooms, the culture solution was collected one day, three days, seven days, and twelve days, and added during MCF-7 cell culture. After 6 days of incubation, the biological activity of the degraded material was measured by the expression of cell proliferation and target genes. As a result, in the case of pore mushrooms, after one day of treatment (FIG. 6), and the large tooth mushrooms after three days of treatment (FIG. 7), cell proliferation was markedly lowered, resulting in decreased bioreactivity.

In addition, the expression level was measured using the pS2 gene, which is a marker gene used in the MCF-7 cell line, and the estrogenity of the degraded compound was verified in parallel with cell proliferation. 8 and 9, when treated with estrogen and the first octylphenol used as a positive reactant, the expression of pS2 gene was induced, whereas it was treated with decomposed products treated with pore mushrooms and mushrooms. In the case of treatment, the expression of pS2 gene in MCF-7 cells was significantly lowered and decreased to the same amount as the untreated group after 1 day of treatment.

According to the present invention, it is excellent in the resolution of alkylphenols, especially octylphenol (4-t-octylphenol), which is used as a surfactant to contaminate water quality and soil. Do.

Claims (2)

Biodegradation method of alkylphenols, which is used as a surfactant and decomposes alkylphenols which pollute the environment, by decomposing singly or mixed strains of big tooth mushrooms or mushrooms in contact with alkylphenols. The method of claim 1, wherein the alkyl phenols are octylphenol (4-t-octylphenol), characterized in that the biological decomposition method of alkyl phenols

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