KR100660389B1 - Biological Recovery Method of Phthalate Compound Using Wood Decay Bacteria - Google Patents

Abstract

The present invention relates to a biological recovery method of the phthalate compound using wood fungi, the biological recovery method of the phthalate compound using wood fungi according to the present invention wood fungi, preferably flowering mushrooms, forest mushrooms, large It relates to a biological recovery method for culturing wood fungi characterized in that at least one selected from the group consisting of bark mushrooms and glue mushrooms to decompose phthalate compounds from the enzymes they produce.

The biological recovery method of the phthalate compound using wood fungi according to the present invention can decompose phthalate compounds corresponding to endocrine disruptors, so in soil or water contaminated with phthalate compounds present in plastic products Useful for biological degradation and repair of

Wood fungus, flowering cloud mushroom, forest mushroom, bark mushroom, glue mushroom, phthalate ester

Description

Bioremediation method of phthalate compound using wood fungus {Bioremediation method of Phthalate Esters by Wood Rot Fungi}

1 shows chemical formulas of diethyl phthalate (DEP), diethylhexyl phthalate (DEHP), dicyclohexyl phthalate (DCP) and benzyl butyl phthalate (BBP) in phthalate compounds.

FIG. 2 shows the resistance of the test strain to diethylphthalate (DEP), diethylhexylphthalate (DEHP), dicyclohexylphthalate (DCP) and benzylbutylphthalate (BBP).

Figure 3 shows the induction effect of laccase and Mn-peroxidase by diethyl phthalate (DEP), diethylhexyl phthalate (DEHP), dicyclohexyl phthalate (DCP) and benzylbutyl phthalate (BBP) .

Figure 4 shows the resolution of the diethyl phthalate (DEP), diethylhexyl phthalate (DEHP), dicyclohexyl phthalate (DCP) and benzyl butyl phthalate (BBP) for each culture period.

5 shows the decomposition mechanism of diethyl phthalate (DEP), diethylhexyl phthalate (DEHP), dicyclohexyl phthalate (DCP) and benzylbutyl phthalate (BBP).

Figure 6 shows the results of cell proliferation for DEP.

Figure 7 shows the results of cell proliferation for DEHP.

Figure 8 shows the results of cell proliferation investigation for DCP.

Figure 9 shows the results of cell proliferation investigation for BBP.

Figure 10 shows the estrogen properties of DCP and its degradation products by the cloud mushroom and forest wrinkle mushroom.

Figure 11 shows the estrogen properties of BBP and its degradation products by the bark mushrooms and glue mushrooms.

The present invention is culturing wood fungi, preferably selected from the group consisting of wood fungi, preferably flowering mushrooms, forest wrinkle mushrooms, bark mushrooms and glue mushrooms phthalate compounds from the enzymes they produce It relates to a biological recovery method to decompose.

Phthalates, which are classified as endocrine barriers, are widely used in the plastics industry, and thus, recently, interest has increased. In Korea, research is mainly conducted on the development of screening methods to determine the priority, monitoring, endocrine disruption, and endocrine disruption in contaminated soil or water quality. The research is very poor.

The use or enhancement of microorganisms for the treatment of contaminated or repellent materials has become a hot topic of recent interest under the generic name bioremediation. Biological studies on the development of endocrine disrupting technologies have mainly focused on bacteria and the like. The research used is dominant. For example, nonylphenol and gaseous styrene can be decomposed using white fungus, or gram-negative aerobic bacteria decompose bisphenol-A. Sphingomonas sp. Has been reported to use nonylphenol as an energy and carbon source.

However, in the degradation of these hardly decomposable substances, research by the microorganisms other than bacteria is not active. Therefore, attention has been focused on the decomposition of hardly decomposable substances such as endocrine disruptors using lignin degrading bacteria and their enzymes having industrial potential. In particular, among the phthalates, dibutylphthalate has been reported to be obtained by decomposing the product into phthalic acid by Pseudomonas pseudoalcaligenes. However, biological degradation using wood fungi, such as white fungi, which degrades lignin, a wood-degradable substance in wood, is insufficient.

Wood fungi secrete peroxidase and polyphenol oxidase, which attack and degrade lignin, a strong polymer, and thus become a major focus of current biological repair methods. The lignin degrading enzyme is a non-specific chlorinated phenol, polychlorinated biphenyl, which is a non-specific recalcitrant compound having a structure similar to that of lignin. polychlorinated biphenyl (PCB), DDT, dioxins, polyaromatic hydrocarbons (PAH), and nitrotoluenes.

However, there is no example developed for the method of decomposing endocrine-caused phthalate compounds by wood fungi, such as flowering mushrooms, forest fungi, bark mushrooms and glue mushrooms, in the present invention, plastic plasticizer among wood fungi By selecting a strain having a resolution to the phosphorus phthalate compound to analyze the resolution and resistance, and to find the effect of reducing the estrogens to establish the decomposition technology for endocrine disruptors to complete the present invention.

An object of the present invention is wood fungus, preferably cloud mushroom, so that it can be used as a biodegradation and recovery method of phthalate compounds in contaminated soil, water quality and biodegradation reactor by spilling out of plasticizer containing phthalate compounds. To provide a biological recovery method for culturing forest wrinkle mushrooms, bark mushrooms and glue mushrooms to decompose phthalate compounds from the enzymes they produce.

In order to achieve the above object, the present invention is characterized by a biological recovery method of culturing wood fungi to decompose phthalate compounds from the enzymes they produce. The wood fungus is preferably characterized in that at least one selected from the group consisting of flowering mushrooms, forest wrinkle mushrooms, bark mushrooms and glue mushrooms.

The present invention relates to a biodegradation and recovery method of phthalate compounds in contaminated soil, water quality and biodegradation reactor in the industry used as plastic plasticizers using phthalate compounds.

      Phthalate compounds, which are plasticizers for plastic products, are endocrine disruptors that show abnormalities in the hormone system and genital organs. The phthalate compounds are preferably phthalate esters. For example, diethylphthalate (DEP), diethylhexylphthalate, DEHP), dicyclohexylphthalate (DCP) and benzylbutylphthalate (BBP), and the like, and the chemical formulas of each are shown in FIG. 1.

The present inventors selected excellent strains showing resistance to phthalate-containing concentrations of 200ppm among 84 species of cultivated strains of the National Forest Research Institute of Microbial Chemistry and 31 kinds of forest mushrooms, Phlebia tremellosa, Polylacticus mushroom (Polyporus brumalis), Flower cloud mushrooms (Stereum hirsutum), forest wrinkle mushrooms (Agaricus silvaticus), bark mushrooms (Lopharia mirabilis), big tooth mushrooms (Basidioradulum molare), and brick beetle butterfly (Heterobasidium insulare) were selected and used as the strain It was.
Where necessary in the specification and drawings of the present invention, the above glue mushrooms are "glue", the winter umbrella is "winter umbrella" or "POB", the flower cloud is "petal cloud", the forest wrinkle is " It is abbreviated as "wood wrinkle" or "forest", big husk mushroom is "big shell" or "LOM", big tooth mushroom is "big teeth", and brick moth butterfly is abbreviated as "brick moth butterfly".

      For each of the target substances, inoculated with the primary starter in malt extract agar (MEA) medium containing 50, 100, 200, 300 and 500 mg / l of control, and incubated at 30 ° C for 10 days. Growth strains were measured for 3, 5, 7, and 10 days to determine the resistance of each strain.

     Figure 2 shows the resistance of the test strains to the phthalate compound, both strains were completely mycelium covered up to 300ppm concentration in DEP and limited growth at 500ppm concentration, but was completely coated after 10 days. DEHP's resistance test showed that the mycelium and winter umbrella were completely covered with mycelia at all concentrations and showed good growth even at high concentrations of 500 ppm. In the DCP, forest mushrooms and flowering mushrooms grew, and in BBP, the growth was limited depending on the concentration. However, both the mushrooms and the bark mushrooms were completely covered with mycelia.

      After investigating the resistance of the four target substances, two strains showing excellent resistance were selected, and the concentration of each target substance was the same in the shallow stationary culture (SSC) medium after the highest mycelial growth. Add to 100 μM in phase and incubated at 30 ℃. In order to investigate the resolution, enzyme titer, and enzyme induction effect of each culture period, the cells were cultured for 1, 4, 7 days without the addition of each target substance, and each target substance was added to the culture medium for 7 days. After incubation for 0, 1, 3, 7, and 12 days, the respective media filtrates were filtered using a 0.45 μm microfilter, followed by titer of manganese peroxidase (MnP), and laccase. Was measured. Titers of MnP 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 and the last addition of the crude enzyme solution initiated the measurement. It was. In the case of laccase titer, only ABTS (0.8 g / L) and 0.1 M sodium lactate buffer were used. The titer of MnP and laccase was calculated by applying ε414 = 36,000M-1cm-1 of ABTS.

3 is a graph showing the induction effect of laccase and Mn-peroxidase by the addition of DEP, DEHP, DCP and BBP. As shown in FIG. 3, laccase and Mn-P showed similar trends in the enzymatic induction effect. Glue mushroom showed a tendency to increase when the target material was BBP, but showed low titer when DEP. Specificity was shown for the material. In the case of DCP, the titer of forest wrinkle mushroom and flowering cloud mushroom increased and decreased immediately after addition.

To determine the resolution of the target material, the filtrate was extracted with hexane and ethylacetate and quantitatively analyzed by HP 1100 series HPLC using a Waters symmetric column to investigate the degradation rate. Analysis conditions were flow rate 0.6ml / min, solvent was CH3CN: water = 80: 20 (v / v) was analyzed under isocratic (isocratic) conditions. Derived solution of hexane and ethylacetate to N, 0-bis (trimethylsilyl) trifluoroacetamide (N, O-bis (trimethylsilyl) trifluoroacetamide) The mixture was reacted at 60 ° C. for 1 hour and analyzed using GC / MS (Shimadzu HiCap-CBP1-M25-025 column, 25 m, 0.25 μm, 0.25 mm).
GC analysis conditions were helium as carrier gas, flow rate 1.0ml / min, injector 270 ℃, detection 285 ℃, oven temperature at 80 ℃ for 5 minutes, 160 ℃ at 8 ℃ / min After rising to 5 minutes, again 5 ℃ / min at 280 ℃ after using the program maintained for 5 minutes was analyzed. The sample (effluent) from the GC column was directly connected to MS, and the spectra were obtained by EI mode, 70 eV ionization energy, and 50 ~ 800 amu scan for 2 seconds. Decomposition product analysis of the obtained spectra was confirmed by comparing with the mass spectra library (Wyley Registry of Mass Spectra Data, 6th ed.) Built in the MS system.

      Figure 4 shows the resolution by the culture period of DEP, DEHP, DCP and BBP. As a result of analyzing the degradation rate by HPLC quantitative analysis of DEP, winter umbrella was 60% degraded, glue was 16% degraded, DEHP was digested 22% at winter umbrella and 49% at cloud mushroom, DCP was flower Both cloud mushrooms and forest mushrooms were decomposed more than 90%, BBP showed 90% degradation of bark mushrooms and 86% of glue mushrooms, indicating that the degradation rate of phthalate compounds by wood microorganisms was very high.

Decomposition mechanisms of DEP and DEHP and DCP analyzed by GC-Mass was the phthalate and ester-bonded portion was cleaved and phthalate was detected as the final product, benzyl alcohol was detected in the case of BBP (Fig. 5).
In this study, the effect of 17β-estradiol, known as an estrogen agonist, on the proliferation of human breast cancer cells, MCF-7 cells, was investigated.
-When cultured MCF-7 cells with DCP, BBP, DEHP, and DEP added at 200 ppm for the first time, the culture medium after 1 day, 3 days, 7 days, 12, 19, 21 days was collected with various mycelia. After 6 days of incubation, the biological activity of the degraded material was measured by the amount of cell proliferation and expression of the target gene.

In order to evaluate the toxicity and safety of the target substance and degradation products, the cell proliferation test was performed by preparing 5% FBS DMEM medium to confirm contamination, and preparing 0.25% trypsin solution and 0.2% EDTA to recover cultured cells, and then performing MCF. After pipetting (Michigan Cancer Foundation) -7 cells, the number of cells was measured. 5% activated carbon was added, distilled water was added to the indicated point, shaken up and down, and the activated carbon was dispersed, followed by centrifugation (250rpm, 2 minutes). The same operation was repeated three more times to remove activated carbon suspended particles. Thereafter, 0.5% dextran (serum amount × 1.1 ml) was added, distilled water was added to the indicated point, and then upside down, 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 in a shaking water bath at 37 ° C. for 60 minutes, centrifuged (3000 rpm, 20 minutes), the supernatant was aspirated and removed with a dropper, and then dispensed into another glass centrifuge tube. The obtained supernatant was filtered through a 0.45 μm filter (3000 rpm, 20 minutes), and 5% of activated carbon treated serum was added to a phenol red free medium for use in an E-screen assay. It was. The cell number was 5 × 10 3 cells / well, and a predetermined amount (100 μl) of cells was dispensed into a 96 well plate (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, followed by removal of the medium in the wells.
90 μl of DMEM containing 5% activated carbon-dextran activated FBS, which is a test medium, was added to each well, and the concentration of the test substance (bisphenol A and methcyclocyclo) was prepared. Was added to the wells. 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 the test substance was added were incubated for 6 days (144 hours) in an incubator with 5% CO2, and the proliferation of MCF-7 cells was induced by the addition of WST-8, a tetrazolium salt. Formazan produced by the reduction reaction was measured at an absorbance of 450 nm.

6 to 9 show the results of examining the cell proliferation of MCF-7 cells for DEP, DEHP, DCP and BBP.

In the case of DEP, the decrease in proliferation by POD was shown to decrease in both stationary and shaking from the 1st day of culture, and it was determined that the glue also had the same effect. E2 is a medium in which estradiol2, a kind of estrogen reagent, is added (FIG. 6).

In the case of DEHP, the proliferative ability was decreased to the level before the addition from the first day of the cultivation by the winter umbrella, and when it was left by the flower cloud, it decreased to the level before the addition from the 7th (FIG. 7).

In the case of DCP, the increase in the post-administration width by the mushroom cloud was reduced to the level before administration at the 19th day, but the growth rate of the cells by the forest wrinkle was decreased after 12 days (FIG. 8).

In the case of BBP, degradation by the bark mushrooms was reduced to the pre-dose level after 3 days of culture. Degradation by glue mushroom was found to decrease to the level before administration on day 21 of culture (FIG. 9).

From the results of cell proliferation experiments on the MCF-7 cells, it can be confirmed that the cytotoxicity is reduced by decomposing each of the target substances into degradation products having reduced cytotoxicity by the present invention.

In addition, the expression level was measured using a marker gene used in the MCF-7 cell line to verify the estrogenity of the degraded compound in parallel with the proliferation of cells. Among the cell proliferative experiments, one of the most presumably effective substances was selected and tested. (Estrogen test subjects were subjected to various tests with DCP, BBP, DEHP, and DEP added at 200 ppm for the first time as in the cell proliferative experiment. 1, 3, 7 days, 12, 19, 21 days after the mycelia were collected and tested).

pS2 mRNA analysis was performed by preparing the cells as in the Proliferation assay, dispensing the calculated cells into 6 well plates at 5 × 10 4 cells per well, incubating for 24 hours in a 5% CO 2, 37 ° C. incubator and then The medium inside was aspirated and removed, and the wells were washed once with 5 ml of PBS, and 2 ml of steroid free DMEM (phenol red-free, DMEM with 5% FBS treated with 5% activated carbon-dextran) was dispensed. . After 72 hours, the steroid-free medium in the plate is treated in the same manner as a Proliferation assay, but two wells per group are designated, and after 24 hours, the medium in the plate is aspirated off and washed once with PBS. Then add 1 ml of Trizol solution (Gibco BRL) per group (2 wells), transfer the pipette solution into a 1.5 ml tube, add 200 ul of chloroform, shake it sufficiently, and then centrifuge (14,000 rpm, 10). Min), remove the supernatant, transfer to a new tube, add isopropanol, invert 4-6 times, centrifuge again (14,000 rpm, 10 minutes), check the pellet, and remove the supernatant with a micropipette. The tube was washed with 70% ethanol (diluted with DEPC treated distilled water), settled in a centrifuge and the remaining ethanol was removed with a micropipette. After adding the appropriate amount of DEPC treated with distilled water (about 15 ul), pipetting to melt the pellet was measured at 600 nm UV, using about 4ug total RNA as a template, using M-MLV reverse transcriptase (Ambion) cDNA was synthesized. PCR was carried out using Tag DNA polymerase as a template of the synthesized cDNA. To amplify the pS2 gene, a forward primer 5'-GGCCACCATGGAGAACAAGG and reverse primer 5'-CCA CGAACGGTGTCGTCGAA were used. 30 times was used. Each cycle undergoes a strain step at 95 ° C. for the first 5 minutes, strained at 95 ° C. for 1 minute, annealed at 50 ° C. for 1 minute, polymerized for 1 minute and 30 seconds at 72 ° C., and expanded ( extension) was repeated 30 times at 72 ° C. for 15 minutes. House keeping gene was used as human 1A, and forward primer 5'-GATATGG CGTTTCCCCGCATA and reverse primer 5'-GGATTTTGGCGT AGGTTTGGT were used for 1A amplification. 50% of the PCR product was loaded on agarose gel (pS-2; 2% agarose, 1A; 1% agarose), and then developed by applying voltage at 100 volts for about 30 minutes and stained using EtBr. Images were analyzed using Gel doc 1,000. The measured pS2 mRNA values were normalized to 1A mRNA.

     Figure 10 shows the estrogen properties of DCP and its degradation products by the cloud mushroom and forest wrinkle mushroom. Expression of pS2 gene was induced by treatment with estrogen used as a positive reactant and the first DCP, but expression of pS2 gene in MCF-7 cells was significantly decreased when treated with degradation products treated with forest wrinkles. Appeared. After 12 days of treatment it was found to decrease by the same amount as the untreated group. However, the decrease was minimal due to the mushroom cloud.

In addition, Figure 11 shows the estrogen properties of BBP and its degradation products by the bark and glue mushrooms, pS2 gene expression was induced when treated with estrogen and the first BBP used as a positive reactant The expression of pS2 gene in MCF-7 cells was significantly decreased when treated with the degradation products of the bark mushrooms. After 19 days of treatment it was found to decrease in the same amount as the untreated group. However, by the 7th day of treatment, the decrease was the same as the administration level.

Therefore, it can be seen from the results of FIGS. 10 and 11 that the estrogen properties of the degradation products of DEP, DEHP, DCP BBP are reduced. As a result, the present invention can be usefully applied to a biological recovery method because the phthalate compound can be decomposed and produced as a safe decomposition product for the human body.

As described in detail above, the present invention can decompose phthalate compounds corresponding to endocrine disruptors, and therefore, the present invention is directed to biological degradation and recovery methods in soil or water and biodegradation reactors contaminated with phthalate compounds present in plastic products. useful.

Claims (5)

Biological recovery method of culturing wood fungi to decompose phthalate compounds from the enzymes they produce. The method of claim 1, wherein the wood fungus is a biological recovery method, characterized in that at least one selected from the group consisting of flowering mushrooms, forest wrinkle mushrooms, large shell mushrooms and glue mushrooms. The biological repair method according to claim 1, wherein the phthalate compound is a phthalate ester. The method of claim 3, wherein the phthalate ester is at least one selected from the group consisting of diethyl phthalate, diethylhexyl phthalate, dicyclohexyl phthalate and benzyl butyl phthalate. The method of claim 1, wherein the enzyme is laccase or manganese peroxidase.

Images