JP3550099B2 - Novel microorganism having dioxin decomposability and dioxin decomposition method - Google Patents

Description

【００３７】

【００３８】
ガスクロマトグラフフィーにおいては、四塩素置換体（ＴｅＣＤＤ及びＴｅＣＤＦ）、五塩素置換体（ＰｅＣＤＤ及びＰｅＣＤＦ）及び六塩素置換体（ＨｘＣＤＤ及びＨｘＣＤＦ）の分析には分離条件Ａ、七塩素置換体（ＨｐＣＤＤ及びＨｐＣＤＦ）及び八塩素置換体（ＯｃＣＤＤ及びＯｃＣＤＦ）の分析には分離条件Ｂを適用して分析を行った。
得られた分析値より、試料中に含まれるダイオキシンのうちの全ての四塩素置換体、五塩素置換体、六塩素置換体、七塩素置換体及び八塩素置換体濃度を、前記のマニュアルに従って算出した。
【００３９】
第１表に飛灰処理試験の試料中のダイオキシン同族体の濃度及びそれらの分解率を、第２表に飛灰処理試験の試料中の毒性等価係数（ＷＨＯ、１９９８年）の示されたダイオキシン異性体の毒性当量及びそれらの分解率の結果をそれぞれ示す。なお、表中のＴＥＱ（Ｔｏｘｉｃ Ｅｑｕｉｖａｌｅｎｔｓ） は毒性当量を指し、ダイオキシン類のそれぞれの異性体の毒性を２， ３， ７， ８−ＴｅＣＤＤに換算して合計したものである。
【００４０】
【表１】
第 １ 表

【００４１】
第１表から明らかなように、本発明の担子菌 ＭＴ００３株をダイオキシン汚染物である焼却飛灰に作用させることによって、該飛灰中に含まれるダイオキシン類を分解、浄化することができる。特に、毒性が最も高いＴｅＣＤＤを４８％、ＴｅＣＤＦを４７％という高率で分解することが明らかとなった。
一般に、ダイオキシンは置換塩素原子数の増加に伴い、分解作用を受け難くなることが知られているが、本発明の方法によれば、四塩素置換体、五塩素置換体、六塩素置換体、七塩素置換体及び八塩素置換体に対して、十分な分解率が得られ、また毒性が高いとされる四塩素置換体、五塩素置換体及び六塩素置換体に対してもＭＴ００３株は高い分解能を有していることが示された。
【００４２】
【表２】
第 ２ 表

【００４３】
第２表は、ダイオキシンの中で毒性等価係数の示されたダイオキシン異性体の飛灰処理試験試料中の濃度を比較したものである。この表から明らかなように、ＭＴ００３株は毒性等価係数の高い四塩素置換体、五塩素置換体及び六塩素置換体に対して、分解率３５〜５２％という値を示し、これらに対する高い分解能を有している。一方、七塩素置換体及び八塩素置換体に対しても、本菌は同様に分解能を有していることが判明した。
【００４４】
【発明の効果】
本発明の担子菌 ＭＴ００３株は、カワラタケ科スポンギペリス属に属する新規な微生物であり、毒性が強く、しかも分解が困難とされているダイオキシン類を分解することができる。特に、毒性の高い四塩素置換体のみならず、４〜８個の塩素原子による置換体の多くに対しても、幅広い分解能を有している。
したがって、この微生物自体、もしくは当該微生物の産生するリグニン分解酵素をダイオキシンにより汚染された焼却灰や土壌等に作用させることによって、これら汚染物を無害化して環境浄化に貢献することができる。
また、本発明の方法は、ダイオキシンの分解を低コストで実施でき、しかも２次廃棄物の発生が少ないという特色を有している。
【００４５】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel microorganism having dioxin decomposability and a method for decomposing dioxin, and more particularly to a novel basidiomycete strain having dioxin decomposability and a method for decomposing dioxin by causing the microorganism to act on dioxin contaminants.
[0002]
[Prior art]
Dioxins, which are organic chlorine compounds, are extremely toxic compounds generated as by-products in improper incineration of waste, production of agricultural chemicals and the like, and chlorine bleaching process of paper and the like.
Generally, the name of dioxin refers to polychlorodibenzo-p-dioxin (hereinafter sometimes abbreviated as PCDD), which is a chlorine-substituted product having dibenzo-p-dioxin as a skeleton, and dibenzofuran as a skeleton. Is a general term for polychlorodibenzofuran (hereinafter sometimes abbreviated as PCDF) which is a chlorine-substituted product.
Since eight hydrogen atoms in the skeleton can be replaced with one to eight chlorine atoms, there are theoretically 210 kinds of compounds in total in dioxins. Its toxicity depends on the position of chlorine substitution and the number of chlorines substituted, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; hereinafter, 2, Is sometimes abbreviated as 3,7,8-TeCDD.), And its acute toxicity is estimated to be 10,000 times that of potassium cyanide.
[0003]
In addition, it has been pointed out that dioxins have not only toxicity but also carcinogenicity and hormone-like action even in a very small amount. Moreover, since dioxins are hardly decomposed by microorganisms or the like, they remain in the environment and are taken into the living body by living organisms together with inhalation and food during respiration.
Furthermore, since dioxins have a property of easily accumulating in living tissues, accumulation of dioxins is more serious in humans at the top of the food chain than in other animals and plants. For this reason, in recent years, environmental pollution by dioxins has become a serious social problem.
[0004]
By the way, as a technique for decomposing and removing dioxins, a method of adding hydrogen peroxide and irradiating ultraviolet rays to decompose it (JP-A-52-035445), and a thermal decomposition method (JP-A-Heisei 6-35445) have been used. 296710), a method for improving combustion conditions (Japanese Patent Publication No. 3-178389), various chemical decomposition methods, a supercritical water treatment method, and the like.
However, it is difficult to apply these known methods to the treatment of dioxin contaminated in a very small amount and over a wide area, and there is a problem that the cost required for the decomposition treatment is generally high. In addition, there is a concern about the generation of secondary waste.
[0005]
On the other hand, attempts have been made to restore the environment by utilizing microorganisms having a high resolution for dioxins. This method has the advantages of low cost and low generation of secondary waste, and is attracting attention as a new environmental purification technology.
Regarding the microbial degradation of hard-to-degrade organic substances, Bumpus et al. Describe a case of decomposition of several kinds of organic substances by Phanerochaete chrysosporium, which is a white rot fungus having the resolution of lignin, which is a hard-to-degrade component of wood. It has been reported. Since this report, there have been reports examining the resolution of many refractory organic substances such as dioxins, mainly white rot fungi (Science, 228, p. 1434, 1985).
[0006]
Enzymes involved in lignin degradation include lignin peroxidase, manganese peroxidase and laccase. It has been found that these enzymes can cooperatively degrade dioxins due to their wide substrate specificity. For example, Valli et al. Decompose 2,7-dichlorodibenzo-p-dioxin (2,7-dichlorodibenzo-p-dioxin, hereafter sometimes abbreviated as 2,7-DCDD) by Fanelokaete chrysosporium. And the involvement of lignin peroxidase and manganese peroxidase produced by the bacteria in the degradation (J. Bacteriology, 174 (7), p. 2131, 1992).
[0007]
Furthermore, it has also been suggested that bacteria capable of producing enzymes involved in lignin degradation generally have the ability to degrade 2,7-DCDD (Paper and Pulp Engineering Co., 50 (12), p. 122). , 1996). In addition, a microorganism having a dioxin decomposability has been newly produced using a cell fusion technique (Japanese Patent Application Laid-Open No. 11-1978).
[0008]
[Problems to be solved by the invention]
As described above, when dioxins are decomposed and removed using a dioxin-degrading microorganism or a dioxin-degrading enzyme produced by the microorganism, they are exhaustively applied to as many as 210 types of dioxins having different chemical structures. It is required to indicate the resolution.
However, the enzyme that decomposes dioxins differs depending on the type of microorganism that produces the enzyme. Therefore, they differ in the basic properties of the enzyme such as substrate specificity, activity or stability.
In addition, for individual dioxin-degrading enzymes, the resolution for each of 210 types of dioxins that are present is not uniform, and there is a difference in the degree of degradation, and enzymes having a wide range of resolution for dioxins are: At present, it has not been found yet.
In particular, the dioxin decomposability of known dioxin-decomposing bacteria mainly focuses on 2,7-DCDD, which is a relatively low-toxic dichlorine-substituted product. The resolving power of the substituted compound with four or more chlorine atoms was not sufficiently shown.
[0009]
A first object of the present invention is to newly provide a microorganism showing a wide range of resolution for various types of dioxins contained in wastes and the like.
Further, a second object is to provide a method for decomposing dioxin using the microorganism or an enzyme produced by the microorganism at low cost and with little generation of secondary waste.
Thus, the present inventors have conducted intensive studies to solve the above-mentioned problems, and have found a novel basidiomycete having lignin decomposability from decaying wood and surrounding soil, and have reached the present invention.
[0010]
[Means for Solving the Problems]
The present invention according to claim 1 relates to a basidiomycete MT003 strain (FERM P-18171) having a dioxin decomposability.
The present invention according to claim 2 relates to a method for decomposing dioxin, which comprises causing the microorganism according to claim 1 or a dioxin degrading enzyme produced by the microorganism to act on dioxin contaminants.
The present invention according to claim 3 relates to the method for decomposing dioxin according to claim 2, wherein the dioxin contaminant is incinerated ash or soil.
The present invention according to claim 4 relates to the method for decomposing dioxin according to claim 3, wherein the incinerated ash is a highly alkaline incinerated ash having a pH of 10 or more.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
A first aspect of the present invention is a basidiomycete MT003 strain (FERM P-18171) having dioxin decomposability. The fungus can be isolated from decayed wood and its surrounding soil using a known method for isolating fungi.
In particular, since this bacterium produces a group of enzymes involved in lignin degradation, for example, by using a medium for separating lignin-degrading microorganisms (Paper and Pulp Technical Co., 50 (12), p. 122, 1996), It can be relatively easily screened as a microorganism. That is, decayed wood pieces collected from the field are suspended in distilled water, and the suspension is appropriately diluted and used as a screening sample. This sample is spread on a separation medium described in the above literature, that is, an agar medium consisting of two layers containing Remazol brilliant blue R in the upper layer, and cultured at an appropriate temperature. Then, a strain capable of decolorizing the dye is regarded as a strain that produces an enzyme involved in lignin degradation, and the colony of the strain is smeared again on the two-layer agar medium, and cultured under the same conditions as described above. By repeating this operation, strains are isolated purely, and strains exhibiting good decolorization results are selected.
[0012]
The basidiomycete MT003 strain according to the present invention is a new species recognized as being classified as white-rot fungi belonging to the family Coriolaceae. That is, the present bacterium was identified by the method described below.
[0013]
According to the method reported by White et al. (PCR protocols, A guide to methods and applications, Academic Press, Inc., New York, pp 315-322, ribosomes of RNA strains of known gene sequences and known ribosomes of RNA strains of known strains). By searching for homology.
First, the MT003 strain is a gene region useful for classification according to the method described in the above-mentioned literature. The 8S ribosomal RNA gene and its nearby 622 base pair base sequence, the 1737 base pair base sequence corresponding to almost the entire length of the 18S ribosomal RNA gene, and the 603 base pair base sequence of the 28S ribosomal RNA gene 5 'end region were determined. The respective nucleotide sequences are shown in SEQ ID NOs: 1, 2, and 3 in the sequence listing.
[0014]
The base sequence of the MT003 strain was determined by the following method. First, freeze-dried cells of the MT003 strain were disrupted in liquid nitrogen, and DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN). Using this DNA as a template, the target ribosomal RNA gene region was amplified by PCR (Polymerase chain reaction). At this time, 5. When amplifying the 8S ribosomal RNA gene region, the primers ITS4 and ITS5 described in the above-mentioned documents are used. For amplifying the 18S ribosomal RNA gene region, the same primers NS1 and NS8 are used. Similarly, PCR was performed using primers LROR and LR7. The amplification reaction of any of the ribosomal RNA gene regions was performed by using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech), preparing four reaction solutions of 40 μmol of primer, 10 to 50 ng of DNA and 25 μl in total of distilled water using GeneAmp PCR. The reaction was performed on a System 9600 (Applied Biosystems). The temperature conditions were as follows: after holding at 96 ° C for 3 minutes, 30 cycles of 94 ° C (30 seconds)-55 ° C (30 seconds)-72 ° C (1 minute), and finally holding at 72 ° C for 4 minutes to terminate the reaction did.
[0015]
Each of the amplified target DNA fragments was subjected to a cycle sequencing reaction by a dye terminator (Dye terminator) method, and the base sequence was analyzed by a DNA sequencer (ABIPRISM 377 DNA Sequencer, Applied Biosystems). The reaction was carried out using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the company's protocol. In the cycle sequencing reaction of each ribosomal RNA gene region, primers and the like described in the above-mentioned literature were used. The base sequences obtained for each amplified fragment were ligated to obtain the target base sequence.
[0016]
For these nucleotide sequences, a homology search of the nucleotide sequences was performed from the GenBank registration data using a BLAST search provided by NCBI (National Center for Biotechnology Information) in the United States.
As a result, it was found that there was no registered strain having the same nucleotide sequence in any of the gene regions in the registered data.
However, 5. Regarding the 8S ribosomal RNA gene, the homology with the gene of Spongipellis directans was 91%, which was the highest in the registered data.
In addition, the 18S ribosomal RNA gene has the highest homology of 98% with Spongipellis unicolor, and in a neighboring binding phylogenetic tree created based on the base sequence of the 18S ribosomal RNA gene of the registered strain, The MT003 strain was found to form a lineage branch with Spongiperis unicolor. Regarding the 28S ribosomal RNA gene, data of the genus Spongipellis has not been registered, but in the above-described phylogenetic tree, Panus radis (Panus rudis), which is closest to Spongiperis unicolor and the registered strain, is not registered. ) Was found to be the highest at 95%.
[0017]
From the above results, it was found that the MT003 strain belongs to the genus Spongipellis of the family Coriolacea, or is a new strain related to the genus Spongipellis. To date, there has been no report that a strain belonging to the genus Spongiperis has the ability to decompose dioxin, and this was first discovered by the present inventors.
The MT003 strain has been deposited with the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, and has a deposit number of FERM P-18171.
The MT003 strain can be inoculated and stored in a commercially available synthetic medium suitable for growth of the present bacterium, for example, a slant prepared using a commercially available potato dextrose agar medium, and used for degradation of dioxins.
[0018]
In the present invention, the term "dioxins" refers to the above-mentioned 210 kinds of PCDD and PCDF. Among PCDDs and PCDFs, those which are particularly problematic are compounds having tetrachloride substitution or higher. In general, it is said that compounds having four or more substituted tetrachlorines are highly toxic and hardly decomposable.
When a specific compound is exemplified, tetrachlorodibenzo-p-dioxin (hereinafter sometimes abbreviated as TeCDD) and tetrachlorodibenzofuran (tetrachlorodibenzofuran; hereinafter, TeCDF) may be used as tetrachloride-substituted compounds. Pentachlorodibenzo-p-dioxin (hereinafter sometimes abbreviated as PeCDD) and pentachlorodibenzofuran (hereinafter sometimes abbreviated as PeCDD). , PeCDF in some cases.).
[0019]
In addition, hexachlorodibenzo-p-dioxin (hereinafter, may be abbreviated as HxCDD) or hexachlorodibenzofuran (hereinafter, may be abbreviated as HxCDF) may be used as a hexachlorine-substituted product. For example, heptachlorodibenzo-p-dioxin (hereinafter may be abbreviated to HpCDD) or heptachlorodibenzofuran (HpCDF may be abbreviated to heptachlorodibenzo-p-dioxin). Octachlorodibenzo-p-dioxin (octachlorodibenzo-p-dioxin). n, hereinafter may be abbreviated as OcCDD) and octachlorodibenzofuran (hereinafter sometimes abbreviated as OcCDF).
[0020]
Next, dioxin contaminants refer to ash and soil contaminated by the above-mentioned dioxins, specifically, incineration ash and fly ash discharged from refuse incinerators, etc. There are emissions from the refining combustion process and the chlorine bleaching process of paper, as well as soil contaminated by these. Among the incinerated ash containing dioxins, there are highly alkaline ones having a pH of 10 or more, particularly 12 to 13. The method of treating microorganisms on such incinerated ash itself to treat dioxins is known as this. According to the present invention, such incineration ash can be treated to decompose and remove dioxins, though not reported so far.
[0021]
Next, a method of decomposing dioxin by causing a microorganism having dioxin decomposability or a dioxin degrading enzyme produced by the microorganism to act on a dioxin contaminant, which is the second invention, will be described.
For the degradation of dioxins, the MT003 strain itself or a dioxin-degrading enzyme produced by the present bacterium is used.
The decomposition of dioxins according to the present invention can be carried out by applying a known method using a microorganism or an enzyme that produces the microorganism. For example, an MT003 strain or a dioxin degrading enzyme produced by the present bacterium is allowed to act on a target containing dioxin contaminants such as incineration ash and soil. More specifically, a nutrient source suitable for the growth of the MT003 strain is added to the dioxin contaminant to be purified, and the MT003 strain is added to the nutrient or a condition suitable for the expression of the enzyme action is added. This is performed by adjusting the temperature (temperature, pH, etc.) and adding the enzyme thereto. The decomposition rate of dioxins can be further increased by changing the type and amount of the nutrient source or by appropriately changing conditions such as temperature, humidity, and pH of the object to be treated during treatment with microorganisms or enzymes. is there.
[0022]
According to the present invention, a dioxin-contaminating microorganism can be decomposed and purified by directly acting on a dioxin contaminant or by applying a dioxin degrading enzyme produced by the microorganism to a dioxin contaminant. . In addition, the MT003 strain according to the present invention has the ability to degrade various types of dioxins, and can also decompose dioxins which are more toxic and harder to decompose than tetrachloride-substituted products. Further, the method of the present invention is lower in cost than the conventional method and generates less secondary waste.
[0023]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not limited thereto.
Example 1 (Isolation of MT003 strain)
In order to isolate a microorganism having a dioxin-degrading ability, the microorganism was separated using a medium for separating lignin-degrading microorganisms (Paper and Pulp Engineering Co., Ltd., 50 (12), p122, 1996). First, decayed wood pieces collected from the field were suspended in sterilized distilled water, and this suspension was used as a sample.
This sample was spread on a medium for separating lignin-degrading microorganisms, that is, an agar medium consisting of two layers containing Remazol brilliant blue R in the upper layer, and cultured at 25 ° C. A colony of the strain from which the pigment was decolorized was picked, smeared on a newly prepared agar medium consisting of the two layers in the same manner, and cultured at 25 ° C. repeatedly, whereby a strain showing lignin degradability was purely isolated. Released.
[0024]
The isolated strain was prepared using a potato dextrose agar medium (per liter, 4.0 g of potato leachate powder, 20.0 g of glucose, 15.0 g of agar, pH 6.0) (manufactured by Nissui Pharmaceutical). (Slope) and cultured at 25 ° C. After confirming the growth of the cells, the slant was stored at 4 ° C.
In this way, a strain showing good decolorization results was isolated. This strain forms white flocculent colonies by culturing on a potato dextrose agar plate medium at 25 ° C. for 5 to 10 days. This bacterium was recognized as a new species belonging to the basidiomycete having the above-mentioned characteristics, and was named MT003 strain.
[0025]
Example 2 (Identification of MT003 strain)
The MT003 strain isolated in Example 1 was identified. The bacterium was identified according to the method reported by White et al. (PCR protocols, a guide to methods and applications, Academic Press, Inc., New York, pp 315-322, 1990) and the ribosome of the MT003 strain of the ribosome of the MT003 gene. The search was performed by searching for homology between the nucleotide sequence and the gene of a known strain.
First, the MT003 strain, which is a useful gene region for classification, The base sequence of 622 base pairs of the 8S ribosomal RNA gene and its vicinity, the base sequence of 1737 base pairs corresponding to almost the entire length of the 18S ribosomal RNA gene, and the base sequence of 603 base pairs of the 5 'end region of the 28S ribosomal RNA gene were determined. .
[0026]
The determination of these nucleotide sequences was performed by the method described above. That is, freeze-dried cells of the MT003 strain were disrupted in liquid nitrogen, and DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN). Using this DNA as a template, the target ribosomal RNA gene region was amplified by PCR (Polymerase chain reaction). At this time, 5. When amplifying the 8S ribosomal RNA gene region, the primers ITS4 and ITS5 described in the above-mentioned documents are used. For amplifying the 18S ribosomal RNA gene region, the same primers NS1 and NS8 are used. Similarly, PCR was performed using primers LROR and LR7. The amplification reaction of any of the ribosomal RNA gene regions was performed by using Ready-To-Go PCR Beads (Amersham Pharmacia Biotech), preparing four reaction solutions of 40 μmol of primer, 10 to 50 ng of DNA and 25 μl in total of distilled water using GeneAmp PCR. The reaction was performed on a System 9600 (Applied Biosystems). The temperature conditions were as follows: after holding at 96 ° C for 3 minutes, 30 cycles of 94 ° C (30 seconds)-55 ° C (30 seconds)-72 ° C (1 minute), and finally holding at 72 ° C for 4 minutes to terminate the reaction did.
[0027]
Each of the amplified target DNA fragments was subjected to a cycle sequencing reaction by a dye terminator (Dye terminator) method, and the base sequence was analyzed by a DNA sequencer (ABIPRISM 377 DNA Sequencer, Applied Biosystems). The reaction was carried out using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the company's protocol. In the cycle sequencing reaction of each ribosomal RNA gene region, primers and the like described in the above-mentioned literature were used. The base sequences obtained for each amplified fragment were ligated to obtain the target base sequence.
The nucleotide sequence of the 5.8S ribosomal RNA gene of the MT003 strain is shown in SEQ ID NO: 1 in the sequence listing, the nucleotide sequence of 1737 nucleotides on the 3 'side of the 18S ribosomal RNA gene is shown in SEQ ID NO: 2, and the vicinity of the 5' side of the 28S ribosomal RNA gene 603 Base configuration of base Column Are described in SEQ ID NO: 3, respectively. In addition, in the base sequence of sequence number 1 of a sequence table, the 33rd-221st base sequence is Internal Transscribed Spacer-1 (ITS-1), and the 383rd-586th base sequence is Internal Transscribed Spacer-2 (ITS-2). ).
[0028]
For these nucleotide sequences, a homology search of the nucleotide sequences was performed from the GenBank registration data using a BLAST search provided by NCBI (National Center for Biotechnology Information) in the United States.
As a result, in the registered data, there was no registered strain having the same nucleotide sequence in any gene region.
However, 5. Regarding the 8S ribosomal RNA gene, the homology with the gene of Spongeperis directans was 91%, which was the highest in the registered data. The 18S ribosomal RNA gene has the highest homology of 98% to Spongiperis unicolor, and the MT003 strain was found to be Spongiperis unicolor in a neighboring phylogenetic tree created based on the base sequence of the 18S ribosomal RNA gene of the registered strain. It was clarified to form a unicolor and a branch.
As for the 28S ribosomal RNA gene, spongiperis genus data was not registered, so it was not possible to search for homology, but it is the closest relative to Spongiperis unicolor and the registered strains in the phylogenetic tree. The homology with Panas ladis was found to be the highest at 95%.
[0029]
From the above results, it was found that the MT003 strain is a bacterium contained in or belonging to the genus Spongiperis of the family Asteraceae, and is a closely related bacterium, and is a new strain. The fact that strains belonging to the genus Spongiperis have the ability to decompose dioxins has not been reported before, and has been found for the first time by the present inventors. The MT003 strain has been deposited with the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, and has a deposit number of FERM P-18171.
[0030]
Example 3 (Production test of lignin-degrading enzyme of MT003 strain)
The lignin-degrading enzyme productivity of the MT003 strain was evaluated by the following method. First, the MT003 strain was collected from the slant stored at 4 ° C., inoculated on a newly prepared potato dextrose agar medium, and cultured at 25 ° C. for 7 days. Next, the cultured cells of one platinum loop were inoculated into 60 ml of a filter-sterilized liquid medium placed in an autoclave-sterilized 300 ml Erlenmeyer flask, and cultured at 25 ° C. for a predetermined period of time. The composition of the liquid medium was 10 g of glucose, 10 g of polypeptone, and 1 g of potassium dihydrogen phosphate per liter. 5 g, magnesium sulfate heptahydrate 0. 5 g, copper sulfate pentahydrate 16 mg and thiamine hydrochloride 2 mg. 6 and used.
The lignin peroxidase, manganese peroxidase and laccase activities in this culture solution were examined daily by the following known measurement methods.
[0031]
Lignin peroxidase activity was measured by a method using veratryl alcohol and hydrogen peroxide as enzyme substrates (Proc. Natl. Acad. Sci. USA, 81, p. 2280-2284, 1983). That is, a solution containing veratryl alcohol (0.4 mM), sodium tartrate (0.1 M, pH 3), and a surfactant (Tween 80, 0.1%) was placed in a cell for measuring absorbance, and further subjected to peroxidation. After adding hydrogen water (0.1 mM), 50 μL of the enzyme was added to carry out the reaction. The reaction was performed at 37 ° C., and the change in absorbance at 310 nm was measured using a spectrophotometer.
The manganese peroxidase activity was measured by a method using divalent manganese ion and hydrogen peroxide as a substrate for the enzyme (Appl. Environ. Microbiol., 57, p2240-2245, 1991). That is, sodium malonate (50 mM), manganese sulfate (0.2 mM), aqueous hydrogen peroxide (0.1 mM) and distilled water were sequentially placed in a cell for measuring absorbance, and 100 μL of the enzyme was added to react. The reaction was performed at 30 ° C., and the change in absorbance at 270 nm was measured using a spectrophotometer.
Laccase activity was measured by a method using syringaldazine as a substrate for the enzyme (Microb. Technol., 3, p. 55-58, 1981). That is, 2-morpholinoethanesulfonic acid-sodium hydroxide buffer solution (0.037 M, pH 5.3), distilled water, and syringaldazine / ethanol solution (0.008 mM) were put into a cell for measuring absorbance, and finally the enzyme was 20 μL was added for reaction. The reaction was performed at 30 ° C., and the change in absorbance at 527 nm was measured using a spectrophotometer.
As a result, it was found that the MT003 strain produced all of the above three types of enzymes involved in lignin degradation extracellularly.
[0032]
Example 4
(1) Test method for dioxin degradation of MT003 strain
The dioxin degrading ability of the MT003 strain was evaluated by allowing the fungus to act on incinerated fly ash and measuring the decomposition rate of dioxins in the ash.
As a dioxin degradability test medium, a mixture of 420 g of sawdust, 60 g of rice bran, and 240 ml of distilled water was used. The mixture was placed in a 6-liter stainless steel container, sterilized in an autoclave (121 ° C., 20 minutes), and then dispersed. Used for ash treatment test.
[0033]
First, one platinum loop of the MT003 strain collected from the stock slant was inoculated into a medium (total amount: 20 g) having the same composition as the above-described dioxin degradation test medium, and precultured at 25 ° C. for 7 days to perform precultured cells. Got. The pre-cultured cells were inoculated on a dioxin degradation test medium in the stainless steel container. This was cultured at 25 ° C. for 14 days, and 120 g of incinerated fly ash whose particle size was adjusted by a sieve was added and mixed well.
After mixing the incinerated fly ash, the mixture was further statically cultured at 25 ° C. for 15 days. As a sample for dioxin analysis, 100 g was weighed immediately after mixing fly ash (day 0) and after culturing for 15 days.
[0034]
(2) Dioxin analysis method
The analysis of dioxin in the sample in (1) above is based on the method described in "Manual for Standard Measurement and Analysis of Dioxins in Waste Disposal" (February 1997) by the Environment Improvement Division, Water Environment Department, Ministry of Health and Welfare. I went. That is, the sample was suspended in 2 mol / L hydrochloric acid, stirred, and filtered. The solid content was washed with water sufficiently washed with hexane (hexane washing water), replaced with methanol, and air-dried. This solid was subjected to Soxhlet extraction with toluene for 20 hours, and the extract was dehydrated with anhydrous sodium sulfate.
[0035]
On the other hand, the filtrate, the washing solution with hexane washing water and the methanol washing solution were subjected to liquid-liquid shaking extraction twice with dichloromethane, and the dichloromethane phase was dehydrated with anhydrous sodium sulfate. The Soxhlet extract and the dichloromethane extract were combined, filtered, and concentrated to 10 mL. Of these, 5 mL was taken and the internal standard was added. Thereafter, the resultant was concentrated and subjected to the following cleanup treatment. That is, the mixture was subjected to multilayer silica gel column chromatography comprising 4 layers of 3 g of silver nitrate silica gel / 0.5 g of silica gel / 4.5 g of sulfuric acid silica gel / 0.5 g of silica gel, and eluted with n-hexane. The eluate was further applied to a column of alumina (6 g), the eluate of n-hexane was discarded beforehand, and dioxins were eluted with n-hexane containing 50% dichloromethane. Further, the eluate was applied to a column of silica gel (0.95 g) embedded in activated carbon, the eluate of 25% dichloromethane-containing n-hexane was discarded beforehand, and dioxins were eluted with toluene. The eluate was concentrated, and a syringe spike was added to obtain a sample for gas chromatography / mass spectrometry. The gas chromatograph mass spectrometry was performed under the following analysis conditions, and dioxin was detected by a selective ion monitoring detection method (SIM detection method).
[0036]

[0037]

[0038]
In gas chromatography, in the analysis of tetrachlorine-substituted products (TeCDD and TeCDF), pentachlorine-substituted products (PeCDD and PeCDF) and hexachlorine-substituted products (HxCDD and HxCDF), separation conditions A, heptachlorine-substituted products (HpCDD and For the analysis of HpCDF) and octachlorine-substituted products (OcCDD and OcCDF), analysis was performed by applying separation condition B.
From the obtained analytical values, the concentration of all tetrachlorinated, pentachlorinated, hexachlorinated, heptachlorinated and octachlorinated dioxins in the dioxins contained in the sample was calculated according to the manual. did.
[0039]
Table 1 shows the concentrations of the dioxin homologues in the fly ash treatment test samples and their decomposition rates, and Table 2 shows the dioxin with the toxic equivalent coefficient (WHO, 1998) in the fly ash treatment test samples. The toxic equivalents of the isomers and the results of their degradation rates are shown respectively. In addition, TEQ (Toxic Equipments) in the table indicates a toxic equivalent, and is a total obtained by converting the toxicity of each isomer of dioxins into 2, 3, 7, 8-TeCDD.
[0040]
[Table 1]
Table 1

[0041]
As is clear from Table 1, the basidiomycete MT003 strain of the present invention acts on incinerated fly ash, which is a dioxin contaminant, to decompose and purify dioxins contained in the fly ash. In particular, it was revealed that TeCDD, which has the highest toxicity, was decomposed at a high rate of 48%, and TeCDF was decomposed at a high rate of 47%.
In general, dioxin is known to be less susceptible to decomposition with an increase in the number of substituted chlorine atoms, but according to the method of the present invention, tetrachloride, pentachloride, hexachloride, The MT003 strain has a sufficient decomposition rate with respect to the heptachlorine-substituted and octachlorine-substituted products, and also has a high MT003 strain with respect to the tetrachlorine-substituted, pentachlorine-substituted and hexachlorine-substituted products which are considered to be highly toxic. It was shown to have resolution.
[0042]
[Table 2]
Table 2

[0043]
Table 2 compares the concentrations of dioxin isomers having a toxic equivalent coefficient among the dioxins in the fly ash treatment test samples. As is clear from this table, the MT003 strain exhibits a degradation rate of 35 to 52% with respect to tetrachlorine-substituted, pentachlorine-substituted, and hexachlorine-substituted products having a high toxicity equivalent coefficient, and has a high resolution for these. Have. On the other hand, it was found that this bacterium also had the ability to degrade heptachloride and octachloride.
[0044]
【The invention's effect】
The basidiomycete MT003 strain of the present invention is a novel microorganism belonging to the genus Spongiperis, belonging to the genus Spongiperis, and is capable of decomposing dioxins that are highly toxic and difficult to decompose. In particular, it has a wide range of resolution not only for highly toxic tetrachloride-substituted products but also for many substituted products with 4 to 8 chlorine atoms.
Therefore, by causing the microorganism itself or a lignin-degrading enzyme produced by the microorganism to act on incinerated ash or soil contaminated with dioxin, these contaminants can be rendered harmless and contribute to environmental purification.
Further, the method of the present invention has a feature that the decomposition of dioxin can be carried out at low cost, and the generation of secondary waste is small.
[0045]
[Sequence list]

Claims (4)

Basidiomycete MT003 strain (FERM) having dioxin resolution
P-18171). A method for decomposing dioxin, which comprises causing the microorganism according to claim 1 or a dioxin degrading enzyme produced by the microorganism to act on dioxin contaminants. 3. The method for decomposing dioxin according to claim 2, wherein the dioxin contaminant is incinerated ash or soil. 4. The method for decomposing dioxin according to claim 3, wherein the incinerated ash is a highly alkaline incinerated ash having a pH of 10 or more.