KR100458000B1 - Bioremediation Method of bisphenol-A and methoxychlor using Stereum hirsutum - Google Patents

Abstract

The present invention relates to a biological recovery method for environmentally harmful substances, and more particularly, to a biological recovery method of culturing flower cloud mushrooms to decompose bisphenol-A or methcyclocyclo from the enzymes they produce.

According to the present invention, it is possible to decompose bisphenol-A corresponding to endocrine obstruction or methoxycyclo, a highly toxic insecticide, and contaminate soil, water waste incinerator, chemicals contaminated with plastic industry and toxic cyclochlorine pesticides. It is useful for microbial recovery methods such as factories.

Description

Biological Recovery of Bisphenol-A and Methoxycyclo Using Flower Clouds {Bioremediation Method of bisphenol-A and methoxychlor using Stereum hirsutum}

The present invention relates to a biological recovery method for environmentally harmful substances, and more particularly, to a biological recovery method of culturing flower cloud mushrooms to decompose bisphenol-A or methcyclocyclo from the enzymes they produce.

Decomposition and estrogen properties of substances classified as endocrine disruptors have been mainly studied using white fungi and bacteria. For example, nonylphenol and gaseous styrene are attempted to decompose using white fungus, gram-negative aerobic bacteria decompose bisphenol-A, and Sphingomonas sp. Uses nonylphenol as energy and carbon source. .

Attention is now focused on lignin degrading bacteria that have industrial potential and techniques using their enzymes. However, in Korea, active research is undertaken only in the areas of priority, monitoring, endocrine disruption measurement method, chemical structure and toxicity evaluation method, and development of screening method to determine endocrine function under the supervision of the Ministry of Environment. There is very little research on technology. In addition, due to the increasing social interest in environmental hormones containing bisphenol-As, the productivity of related industries is extremely weakened. Therefore, the establishment of decomposition technology for these harmful substances is urgently needed.

The process of using or enhancing microorganisms for the treatment of contaminated or repellent materials has become a focus of recent interest under the generic name bioremediation. Wood lysing bacteria secrete peroxidase and polyphenol oxidase, which attack and degrade lignin, a strong polymer, and thus are the main focus of current biological repair methods. Lignin degrading enzymes are non-degradable compounds that are non-specific in terms of polymer degradation and have a lignin-like structure. It is possible to decompose toluene and the like.

The present inventors have completed the present invention by confirming that the flowering cloud mushroom among the wood degradation bacteria has excellent resolution and resistance against bisphenol-A classified as an endocrine disruptor and methcyclocyclo which is a highly toxic insecticide. Was done.

As described above, an object of the present invention is to provide a method for decomposing bisphenol-A or methcyclocyclo from enzymes secreted by the flower cloud mushroom.

1 is a graph showing the results of measurement of the resistance of the flower cloud mushroom to bisphenol-A

Figure 2 is a graph of the results of measurement of the resistance of the cloudiness mushroom to methcyclocyclo

Figure 3 HPLC chromatogram (b) after incubation for 7 days in bisphenol-A standard (a) and flowering mushroom

Figure 4 is an HPLC chromatogram (b) after culturing for 1 day in the methcyclocyclo standard (a) and flower cloud mushroom

5 is a graph showing the results of measuring the resolution of each bisphenol-A culture period by flowering cloud mushroom

6 is a graph of mycelial growth according to the temperature and culture period of the flowering cloud mushroom

7 is a graph showing the change in enzyme titer according to the content of carbon source and nitrogen source

8 is a graph showing the results of cell proliferation of bisphenol-A products decomposed by the flower cloud mushroom

9 is a graph showing the results of measuring cell proliferation of methcyclocyclo product decomposed by the flower cloud mushroom.

FIG. 10 is an electrophoresis photograph of the effect of pS2 gene expression by bisphenol-A products decomposed by the flower cloud mushroom.

FIG. 11 is an electrophoretic photograph of the effect of pS2 gene on methcyclocyclo product decomposed by cauliflower mushroom.

12 is a photograph of the effect of bisphenol-A concentration on immature rat uterine tissue

The present invention for achieving the above object includes a biological recovery method of culturing the flower cloud mushrooms to decompose bisphenol-A or methcyclocyclo from the enzymes they produce.

Flower cloud mushroom ( Stereum hirsutum ) is distributed throughout the world in Korea, including Valwangsan , Jirisan, Manduksan, Hallasan, Duryun, and Japan, and its occurrence is a wood debris that grows and decomposes on dead wood or shiitake trees all year round. It is classified as white fungus that can break down lignin into bacteria.

According to the present invention, these flower cloud mushrooms are used as a coating agent such as metal tubes and have the ability to decompose bisphenol-A, which is classified as an endocrine barrier, or methoxycyclo, a highly toxic insecticide, and at the same time, resistance to these substances It can be confirmed that excellent. In addition, as a result of the experiment, the effect of reducing the estrogen as well as the toxicity test in the animal was confirmed that it is not toxic, confirming that it can be widely used in biological recovery methods.

The cloud mushroom does not require limitation to a particular strain. Therefore, it should be noted that any strain is applicable to the present invention. Therefore, the purchase of the strain can be obtained by using a commercial route that is well known to those skilled in the art, can be obtained directly from nature.

Typical examples of where bisphenol-A or / and methoxycyclo are problematic are the plastics industry, soil contaminated with toxic cyclochlorine pesticides, water waste incinerators, chemical plants and the like. The treatment of these harmful substances can be carried out by mediating the enzyme reaction by culturing the mushrooms by forming the same culture conditions as in Example 2 in a bioreactor (Bioreactor) and the like. However, such a treatment method is merely exemplary for describing the present invention, and those skilled in the art will be able to devise various methods as necessary.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples.

<Example 1>

YMPG medium containing 50, 100 and 500 mg / l of bisphenol-A and methcyclocyclo (10 g glucose per liter of distilled water, 10 g malt extract, 2 g bacto-peptone, 2 g yeast extract, 1 g asparagine, 1 g KH ₂ PO ₄₎ , MgSO ₄ 7H ₂ O 1g, thiamine 1mg, bacto-agar 20g) was inoculated with flowering mushrooms and cultured at 30 ° C. for 14 days to measure the growth length of the mycelia growing daily to determine the resistance of the strain.

In addition, after the highest mycelial growth of the strains grown on YMPG medium, 50 μl from 20mM stock solution of bisphenol-A and methoxycyclo were added to the flask containing 10 ml of SSC liquid medium, and the concentration of the compound in the liquid medium was 100 μM. Adjusted to incubation at 30 ° C.

After incubation for 1, 3, 7, 14 days to examine the resolution of each culture period, 20 ml of hexane was added, shaken vigorously for 5 minutes, left for 10 minutes, and 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, filtered using a 0.45 ㎛ microfilter, and quantified by HP 1100 series HPLC using a Waters symmetric column. The detection was carried out at UV 280 nm. In the case of methcyclocyclo, the analysis conditions were 0.8 ml / min flow rate, the solvent was CH ₃ CN: water = 95: 5 (v / v), the bisphenol-A was 0.6 ml / min flow rate, and the solvent was CH ₃ CN: Water was analyzed under isocratic conditions of 80:20 (v / v).

The results of measuring the growth cycle of mycelia for 14 days by inoculating a cloud of mushrooms in a medium containing bisphenol-A and methcyclocyclo 50, 100, and 500 ppm were as in FIGS. 1 and 2.

When the mycelium completely covered the chalet and the growth ring was 8.5 cm, it was regarded as full growth. First, the results shown in FIG. 2 showed that the control group of the cloudiness mushroom (group without addition of bisphenol-A) took 6 days to complete growth, and the maximum mycelial growth rate was 2.5 cm / day, and average 2 cm / day. However, in the 50ppm bisphenol-A containing medium, the flowering cloud took 7 days, and the growth was slightly inhibited with an average growth rate of 1.5 cm / day. Even at 100ppm, flowering cloud mushroom took 8 days, and mycelial growth was not significantly inhibited by bisphenol-A addition up to 100ppm. However, it was found that at 500 ppm, which is a high concentration, no growth occurred, and 500 ppm completely inhibited mycelial growth.

In the medium containing 100 ppm concentration of methoxylcyclo, flowering cloud mushrooms took 8 days to delay the growth of 2-4 days than the control group. However, unlike the bisphenol, the mycelial growth of the strain was weakly achieved even at over 500 ppm. Could see.

As a result of investigating the resolution of the target material using HPLC, the peak of the bisphenol-A standard was confirmed to have a retention time of 4.924 minutes at 280 nm in the chromatogram at the bottom of FIG. 3 (a), and (b) inoculated with a cloud mushroom. HPLC chromatograms of degradation products incubated for 7 days showed peaks of various degradation products of bisphenol-A.

The peak for the methcyclocyclo standard was found to have a retention time of 4.854 minutes in Fig. 4 chromatogram (a), and the chromatogram (b) was obtained from the degradation products incubated for 1 day after adding methcyclocyclo to the flowering cloud mushroom. It appeared to be completely degraded as a peak. In addition, all the samples incubated for 3, 7, and 14 days could not find a peak for methoxylcyclo, and only peaks related to degradation products existed.

As a result of investigating the resolution of the flowering cloud mushroom of bisphenol-A is as shown in Figure 5, the initial concentration of 200ppm was 63.1ppm on the 1st day, 45.6ppm on the 3rd day, 77% was decomposed and the trace remains on the 7th day, the 14th is complete It was found to decompose.

Example 2 Culture Conditions for Maximizing Enzyme Production

The mycelial growth was measured by inoculating at 2% maltextract agar and YMPG medium to incubate at 17, 25, 30, and 34 ° C., respectively, to select an appropriate temperature and medium for the flowering mushrooms. In addition, in order to measure the change in enzyme titer according to carbon source and nitrogen source, glucose and Ammonium nitrate medium contents were adjusted and cultured in SSC medium, and then the titer was measured by filtration of crude enzyme solution at the time of mycelial growth.

Enzyme titers of manganese peroxidase (MnP) and 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) solution, 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 titer of MnP and laccases is that of ABTS ₄₁₄ = 36,000 M ^-1 cm ^-1 was calculated by applying. In addition, in order to observe the titers of MnP and laccase enzymes changed by bisphenol-A, the titer was examined by the above method at intervals of 1, 3, 5, 7, 9 days after inoculating the strain into SSC liquid medium.

As a result of comparing the mycelial growth while changing the medium and temperature is shown in FIG. The cloud mushroom showed a marked inhibition of mycelial growth at 17 ° C, but 30 ° C was the optimal temperature at 25, 30, and 34 ° C, although there was a slight difference in growth rate. The YMPG medium showed better mycelial growth than the 2% malt extracted agar medium at the same temperature. Therefore, it was determined that the optimum growth was reached in YMPG medium at 30 ℃.

The results of examining changes in enzyme titers according to carbon and nitrogen sources are shown in FIG. 7. In the cloud mushroom, MnP and laccase titers reached the maximum when the nitrogen concentration of ammonium tartrate was doubled, but there was no improvement in enzyme activity due to the increase in the concentration of carbon (glucose).

Example 3 Estrogen Reduction Effect on Target Substances and Degradation Products

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

The medium of the 25 cm ³ flask containing the cells was aspirated / removed for the passage of cells, and then washed with 5 to 6 ml of PBS buffer to remove the residues remaining on the bottom surface. After that, 500% of the 0.25% trypsin solution was added to the flask and incubated for 1 to 2 minutes. When MCF-7 cells were separated from the bottom of the flask, 5 ml of medium was pipetted into the flask, and the cells were dispensed into a 15 ml centrifuge tube and centrifuged (1000 rpm, 4 ° C., 5 minutes). Supernatant was aspirated / removed with an aspirator, fresh medium was added, pipetted to unify cells, and cell number was measured. New 25cm 5㎖ into a 5% FBS DMEM media in flask ^3, 4~5 × 10 ⁴ cells / busy the cell so that the flask and then the flask was shaken to ensure that the cells are uniformly dispersed slowly 5% CO _2, 37 ℃ incubator Incubated at. Once every 3-4 days, the medium was aspirated / removed with aspirator and replaced with fresh medium. Cell passage was carried out at 6-8 days intervals.

Activated carbon-dextran treatment was performed to remove the cell growth factor contained in the serum. Distilled water with a serum volume of 1.1 ml was placed in a glass centrifuge tube with a lid, and the distilled water was removed after marking the surface accurately. 5% activated carbon (serum amount × 1.1 ml) was added thereto, 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. Thereafter, 0.5% dextran (serum amount × 1.1 ml) was added, distilled water was added to the indicated point, and then turned upside down to disperse activated carbon and centrifuged (600 rpm, 5 minutes). After inhaling / removing the supernatant, serum was added and the mixture was slowly stirred to prevent foaming and activated carbon was dispersed. This was incubated for 60 minutes in a shaking water bath at 37 ℃ centrifuged (3000rpm, 20 minutes). The supernatant was aspirated / removed with an eyedropper and dispensed into another glass centrifuge tube, followed by centrifugation again (3000 rpm, 20 minutes). The supernatant obtained by repeating the above procedure twice was filtered with a 0.45㎛ 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 the activated carbon treated serum to phenol red free medium was used for the E-screen assay.

In the cell proliferation test, one day before dispensing the cells into a 96 well culture plate, the medium of the cells to be used for the test was exchanged, the medium in the flask containing the cells was removed on the day of the test, washed once with PBS buffer, and then 0.25%. Trypsin was incubated for 1-2 minutes with the addition of 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 by an aspirator. 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 hemocytometer. The cell number was 5 × 10 ³ cells / well, and a predetermined amount (100 μl) of cells was dispensed into a 96 well plate. After slowly shaking the 96 well plate to uniformly disperse the cells and incubate for 24 hours in a 37 ℃ incubator in which 5% CO ₂ is kept constant, the medium contained in the wells was removed. 90 μl of DMEM containing FBS activated with 5% activated carbon-dextran, a test medium, was added to each well, and the concentration of the test substance (bisphenol-A and methoxycyclo) was prepared and 10 μl was added to each well. . 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 substance was added were incubated for 6 days (144 hours) in an incubator at 5% CO ₂ , MCF-7 cells were grown, and total RNA was isolated and the expression level of pS2 gene was determined by RT-PCR method. Measured.

pS-2 mRNA analysis was performed by preparing the cells as in the cell proliferation test, and the calculated cells were dispensed into 6 well plates to be 5X10 ⁴ cells per well, and then incubated in a 5% CO ₂ , 37 ° C incubator for 24 hours. After 24 hours, aspirate / remove the medium in the well and wash the well once with 5 ml of PBS, and 2 ml of steroid-free DMEM (phenol-free DMEM with 5% FBS treated with 5% activated carbon-dextran) without steroids Each was busy. After 72 hours, the steroid-free medium in the plate was treated by the same method as the proliferation assay, but two wells were designated per group. After 24 hours, the medium in the plate was aspirated / removed, washed once with PBS, and then 1 ml of Trizol solution (Gibco BRL) was added to each 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, turn it over 4 to 6 times, and then turn it over again. Centrifugation (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 distilled water), precipitated by centrifuge, and the remaining ethanol was removed with a micropipette. The pellet was dissolved by adding distilled water treated with an appropriate amount of DEPC (about 15 µl) and pipetting. The concentration was measured at 600 nm of ultraviolet light, and about 4 μg of total RNA was used as a template, and cDNA was synthesized using M-MLV reverse transcriptase (Ambion). When PCR was performed using Tag DNA polymerase as a template with 1 μl of the synthesized cDNA, 30 times was performed using a ford primer 5'-GGCCACCATGGAGAACAAGG and reverse primer 5'-CCA CGAACGGTGTCGTCGAA to amplify the pS-2 gene. Was performed. Each cycle was subjected to a strain step at 95 ° C. for the first 5 minutes, strained at 95 ° C. for 1 minute, annealing at 50 ° C. for 1 minute, polymerization at 72 ° C. for 1 minute 30 seconds, and extension repeated 30 times at 72 ° C. for 15 minutes. The housekeeping gene was used as human 1A, and the four primer 5'-GATATGG CGTTTCCCCGCATA and the reverse primer 5'-GGATTTTGGCGT AGGTTTGGT were used to amplify IA. 50% of the PCR product was loaded on agarose gel (pS-2; 2% agarose, 1A; 1% agarose) and run 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 examined using 17β-estradiol, which is known as an estrogen agonist, as a positive control (FIG. 8). In the case of bisphenol-A, the proliferation of the cells at the concentration of the first 200 ppm was reduced by 40% after one day of treatment by the flower cloud, and after 3 days, it showed the same reaction as the untreated group. Is estimated to decrease in biological activity after 3 days.

In the case of methcyclocyclo, the biological reactivity of the degradation products was completely reduced by the treatment of the cloud mushroom, and it was shown that methoxycyclo was more sensitive to the mycelium than these bisphenol-A (FIG. 9).

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. As shown in FIG. 10, pS2 gene expression was induced when treated with estrogen and BPA used as a positive reactant, but expression of pS2 gene in MCF-7 cells when treated with degradation products treated with flowering clouds Was found to decrease significantly. It was observed in each strain that persisted with a decrease of about 30% or more after 1 day of treatment.

As with bisphenol-A, the expression level of pS2 used as a marker gene in MCF-7 cell line treated with methoxycyclo was measured to verify the estrogenity of the degraded compound in parallel with cell proliferation. As shown in FIG. 11, pS2 gene expression was induced when treated with estrogen used as a positive reactant and the first methcyclocyclo, but was treated with pS2 of MCF-7 cells when treated with lysate treated with a cloud cloud. Gene expression has been shown to decrease significantly after the last day of treatment. About 1 day after treatment, only the medium was found to decrease to the same level as the added group.

As a result of analyzing bisphenol-A in immature rats, the effect on tissues was observed, especially in the uterus. Significantly increased proliferation of uterine gland and epithelial cells with concentration (FIG. 12). However, in the case of methcyclocyclo, it was a weak level.

According to the present invention, it is possible to decompose bisphenol-A corresponding to endocrine obstruction or methoxycyclo, a highly toxic insecticide, and contaminate soil, water waste incinerator, chemicals contaminated with plastic industry and toxic cyclochlorine pesticides. It is useful for microbial recovery methods such as factories.

Claims (1)

Biological recovery method of culturing cauliflower mushrooms to break down bisphenol-A or methcyclocyclo from enzymes produced by them