JP2003052367A - Method of decomposition treatment for organic contaminant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and rapidly decomposing organic contaminants in an organic solvent by using laccase or the laccase together with a laccase mediator. SOLUTION: This method of decomposition treatment for organic contaminants is to carry out the decomposition treatment for the organic contaminants by using the laccase included in reversed micelles in a condition with adding the laccase mediator or without adding the laccase mediator.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for decomposing an organic pollutant, and more particularly to a method for decomposing an organic pollutant using an enzyme in an organic solvent.

[0002]

2. Description of the Related Art Chlorophenols, polychlorophenols, dioxins, PCBs (polychlorinated biphenyls), DDTs (dichlorodiphenyltrichloroethanes), HCBs (hexa) are generally known as organic compound-based environmental pollutants, so-called organic pollutants. Chlorobenzene), T
In addition to organic halogen compounds such as CE (trichloroethylene) and PBBE (polybromobiphenyl ether), alkylphenols, bisphenol A, phthalates, DES (diethylstilbestrol), organotin compounds, some organochlorine compounds, etc. Endocrine disrupting chemicals (environmental hormones), azo compounds used in various dyes and detergents, polycyclic aromatic hydrocarbons (PAHs), and the like. These compounds are
It is generally biodegradable and has been confirmed to remain after being released into the environment, and some of these compounds have been found to adversely affect the ecosystem. However, decomposition treatment of many of these compounds has not been established, and establishment of a safe treatment method is an urgent issue.

Typical examples of the hardly biodegradable organic pollutants are organic halogen compounds. Of these, various organic chlorine compounds that have been conventionally used for applications such as pesticides, insulators and preservatives are generally sparingly soluble or insoluble in water, so biological treatment by dissolving them in water is impossible. It is said that Since these are excellent in stability, many of them remain in the environment for a long time. In particular, it has been revealed that chlorophenols and chlorobenzenes produce extremely toxic dioxins due to incomplete combustion at low temperatures. Therefore, in the complete decomposition treatment of the organic chlorine compound, a treatment of combusting and decomposing with a fuel is performed in a high-temperature combustion furnace specializing in hazardous waste under strict pollution regulations. However, since the treatment is performed in a high-temperature incinerator, (i) a large amount of fuel is consumed, (ii) a high-temperature incinerator specialized in hazardous waste is large-scale, (ii)
There are problems such as i) the need for a cooling device and (iv) the need to transport the product to an incinerator, and the cost is enormous as a whole. In addition, it is necessary to continue monitoring for the production of toxic compounds including dioxins during the incineration of organochlorine compounds. Furthermore, it cannot be denied that the chemical substances produced after combustion may contain unpredictable and unknown toxic compounds. Due to such various circumstances, it is difficult to obtain social consent regarding the construction of such an incinerator.

Under these circumstances, several treatment methods have been devised in place of the commercialized high temperature incineration method. For example,
As physicochemical treatment methods, X-ray irradiation, ozone contact, activated carbon adsorption, supercritical reaction, and other methods have been implemented or investigated, but they are effective in decomposing organic chlorine compounds released and diffused into the environment. I can't say that. Therefore, some biodegradation treatment methods using microorganisms or enzymes produced by them have been devised (JP 2001-46052A, JP 11-309443A, JP 10-257895A, JP 6-912290A). However, there is room for consideration and it has not been put to practical use. The reason is that the decomposition treatment is inefficient because the decomposition treatment is performed in an aqueous system in which the organochlorine compound is difficult to dissolve. It can be limited.

Here, laccase (EC1.10.3.2), which belongs to oxidoreductase, is an enzyme that catalyzes one-electron oxidation of various substrates by using molecular oxygen under mild conditions and is substrate-specific. The property is not critical, and it has high reactivity with aromatic compounds. Further, among the laccase substrates, a low-molecular compound that becomes a relatively stable radical ion after being oxidized is called a laccase mediator. It was revealed that laccase mediators radically ionized by laccase act on a group of hardly biodegradable organic pollutants that are usually difficult to decompose or cannot be decomposed by laccase alone, and indirectly decompose these organic pollutants. (Bressler, D .;
C. et al . : Biotechnol. Let
t. , 22, 119-125 (2000); Srebo.
tnik, E .; et al . : J. Biotechno
l. , 81, 179-188 (2000)).

Most of the studies on the decomposition of organic pollutants by using these laccases alone or the combined use system of laccase and laccase mediator (laccase-laccase mediator system) are related to treatment in an aqueous solution system, and organic pollutants are generally used. Considering that it is highly hydrophobic, it is hard to say that it is a suitable treatment method for decomposing organic pollutants. The same is true for known patents dealing with the laccase-laccase mediator system. For example, "dioxin decomposition method"
(Japanese Patent Laid-Open No. 2001-37465) is basically performed in an aqueous solution, and the decomposition efficiency is poor. Even if the method described in this patent is carried out in an organic solution, it is self-evident that the enzyme is rapidly inactivated and denatured.

[0007] In recent years, regarding the decomposition treatment of organic pollutants, the method of decomposing after concentrating and extracting with an organic solvent is low in cost, and it is easy to handle from the inconvenient microbial treatment method because live cells are used. Researchers at universities and companies agree that the flow of research is changing to a certain enzyme method. In such a situation, as a general-purpose method for decomposing an organic pollutant, which is an alternative to the incineration method and does not cost much energy, a decomposing method applying a laccase-laccase-mediator system in an organic solution is partially related. Has been sought after by people. However, at present, such a decomposition treatment method is not known.

[0008]

The present invention provides a method for effectively decomposing various organic pollutants present in organic solvents by using a laccase or a laccase-laccase mediator system. It is an object of the present invention to provide a method of dechlorinating an organic solution containing an organic chlorine compound when decomposing the organic chlorine compound and reducing the total amount of the remaining organic chlorine.

[0009]

As described above, in order to efficiently decompose organic pollutants such as chlorophenols, bisphenol A, PCB, dioxins and PAHs at a high concentration, it is necessary to use an organic solvent. Required. However, regarding the functional expression of laccase in an organic solvent,
When a conventional method is applied, it is rapidly or gradually denatured / inactivated, and so far no effective method for expressing a function has been known.

In order to solve the above-mentioned problems, the inventors of the present invention have made extensive studies on the development of a general-purpose decomposition treatment method for the above organic pollutants using laccase. The present inventors investigated an environment in which the function of laccase can be expressed in non-aqueous and water-containing organic solvents in order to allow laccase to efficiently act on various organic pollutants. The present inventors can further enhance and transfer the oxidative power of laccase indirectly by incorporating the laccase-mediator treatment system after setting the environment so that laccase can stably function in an organic solvent. Therefore, it was thought that laccase alone can efficiently decompose even target compounds that are difficult or impossible to decompose.

As a result, laccase is stabilized by encapsulating laccase in reverse micelles formed by a certain kind of surfactant, and this entrapping laccase rapidly dissolves organic pollutants dissolved in an organic solvent. Disassemble into
The inventors have found that the combined use of laccase and laccase and mediator enhances the decomposition efficiency, and have completed the present invention.

[0012] In one aspect, the present invention provides a method for expressing laccase activity in an organic solvent, which comprises the step of incorporating a laccase-containing substance into reverse micelles by using a surfactant involved in reverse micelle formation. provide.

In one embodiment, the method may include the step of using the reverse micelle in the presence of a laccase mediator content, which may enhance and transfer laccase activity in an organic solvent.

In another embodiment, the organic solvent may include an organic contaminant, which may be decomposed.

In another embodiment, the surfactant may be sodium di-2-ethylhexyl sulfosuccinate or cetyltrimethylammonium bromide.

In another embodiment, the organic solvent is
It may be insoluble or sparingly soluble in water.

In another embodiment, the organic solvent may be isooctane.

In another embodiment, the laccase
The mediator-containing material can be a 1-hydroxybenzotriazole-containing material.

In another embodiment, the organic pollutant may be composed of a chemical selected from the group consisting of organic halogen compounds, endocrine disrupting chemicals, azo compounds, and polycyclic aromatic hydrocarbons.

In another embodiment, the above method is characterized in that the organic pollutant is o -chlorophenol, m -chlorophenol, p -chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol. , 2,
It may be composed of a chemical selected from the group consisting of 4,5-trichlorophenol, and bisphenol A.

[0021] In another embodiment, the laccase-containing material may be a culture of a laccase-producing bacterium or a processed product thereof. In a preferred embodiment, the laccase-producing bacterium is Trametes s.
p. ) Ha1 strain. In another preferred embodiment, the laccase-producing bacterium is selected from the group consisting of a strain belonging to the subphylum Zygomycete, a strain belonging to the subphylum Ascomycete, a strain belonging to the subdivision Basidiomycete and a strain belonging to the subphylum Incomplete. Can be done. In a further preferred embodiment, the laccase-producing bacterium is Mucor , Neurospora , Podospora .
ora ), Coryvia ( Colybia ), Fome ( Fomes ), Lentinus ( Lentinus ), Schizophyllum , Trametes , Coriolus ( Coriolu )
s), Sanateforasu (Thanatephoru
s), Rhizoctonia (Rhizoctonia), Coprinus (Coprinus), Pusachirera (Psaty
rella ), polyporus ( Polyporus ), Pycnoporus ( Pycnoporus ), phlevia ( Ph )
lebia ), Hygrophorsis ( Hygrophor )
opsis), Agaricus (Agaricus), Basuserumu (Vascellum), Kurushiburumu (Cru
cibulum), Supororumiera (Sporormi
ella ), Pleurotus ,
Gurifora (Grifora), Ganoderma (Gano
derma), Renjitesu (Lenzites), Fanerokete (Phanerochaete), Seri poly Opsys (Ceriporiopsis), Rigidoporasu (Rigidoporus), Fomitera (Fomi
tella ), Trachyderm
a), Aspergillus (Aspergillus), Alternaria (Alternaria), Botrytis (Botrytis), Nectria (Nectri
a), Myrothecium (Myrothecium), Sera toss file area (Ceratosphaeria), Sutakiridiumu (Stachylidium), Sutirubera (Stilbella), Sagenomera (Sage
Nomella ), Pyricularia ( Pyricula )
ria ), Mycelio phtor
a ), citaridium ( Schytalidium ), heterobasidium ( Heterobasidium ),
Fiforoma (Hypholoma), Reputoporasu (Leptoporus), Panas (Panus), Sutereumu (Stereum), Roserinia (Rosel
linia ), Fomitopsis ( Fomitopsi )
s), it may be selected from the group consisting selected from strains classified into the genus Foriota (pholiota) and Acremonium (Acremonium). In an even more preferred embodiment, the laccase-producing bacterium is Trametes villo.
sa ), Miseriofutora Thermophila ( Mycel)
iophthora thermophila ), Citalium thermo film ( Scytalidium)
thermophilum), Rhizoctonia solani (Rhizoctoniasolani), Myrothecium Vel career (Myrothecium ver
rucaria), Myrothecium Roridamu (Myro
therium roridum ), Hitotake mushroom ( Co
prinus cinereus ), oyster mushroom ( Py
Cnoporus coccineus ), Coriolus versicolor , Coriolus hirsutus ,
Suehiro bamboo ( Schizophyllum comm
une ), Pleurotus ostre
atus ), Bekko ( Fomitella fr )
axinea), Pyricularia oryzae (Pyri
cularia oryzae ), Botrytis cinerea ( Botrytis cinerea), shrimp ( T )
rachyderma tsunodae ), sulmetake ( Rigidoporus zonalis ), Aspergillus awamori ( Aspergillus aw)
amori ), Aspergillus nidulans ( Asp
ergillus nidulans ), Aspergillus niger ( Aspergillus niger ),
Aspergillus
oryzae ), Aspergillus terreus ( Aspe
rgillus terreus ), Aspergillus
Soyae ( Aspergillus sojae ), Acremonium Murorum ( Acremonium mur)
orum ) and variants thereof.

In another embodiment, the laccase-containing material entrapped in reverse micelles by sodium di-2-ethylhexyl sulfosuccinate has the following degradation characteristics for o -chlorophenol dissolved in isooctane: 1) Stabilizing pH range: pH 3.0 to 8.0;
(2) Stabilizing temperature range: 40 ° C. to 75 ° C .;

In one aspect, the present invention is a method of degrading organic contaminants, the method comprising: mixing a laccase-containing material with an organic solvent containing a surfactant to form reverse micelles containing laccase. And a step of causing the reverse micelle to act on an organic pollutant.

In one embodiment, the above method comprises
The method may further include the step of mixing the laccase mediator content.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION The present application relates to a method for expressing laccase activity in an organic solvent, which comprises a step of incorporating a laccase-containing substance into reverse micelles using a surfactant involved in reverse micelle formation. In one embodiment, the method can include the step of using reverse micelles in the presence of laccase mediator containing. Furthermore, the laccase activity expressed in organic solvents can decompose organic pollutants in organic solvents.

(Laccase) The laccase used in the present invention has an activity of catalyzing the one-electron oxidation of various substrates using molecular oxygen (“laccase activity”), and its substrate specificity is It is an enzyme that is not strict and has high reactivity with aromatic compounds. Such a laccase is a laccase derived from a microorganism or an animal or plant. Preferably, it is derived from a microorganism. Microorganisms that produce laccase (hereinafter "laccase-producing bacteria")
May be a fungus, in particular a strain belonging to the subphylum Zygomycete, a strain belonging to the subphylum Ascomycete, a strain belonging to the subphylum Basidiomycete or a strain belonging to the subphylum Incomplete. Not limited to. As laccase-producing bacteria, for example, lignin-degrading wood rotting fungi, are particularly well known white-rot fungi belonging to Basidiomycetes, in particular, Hourokutake genus (Tra
metes sp. ), Coriolus genus (Coriolu
s sp. ), Pycnoporus
sp. ) And so on. In addition, the laccase-producing bacteria of the present invention include Mucor ( Muco
r), Neurospora (Neurospora), Podosupora (Podospora), Koribia (Colly
bia ), Fomesu ( Fomes ), Lentinas ( Le
ntinus ), Schizophylle
lum ), Trametes ( Trametes ), Coriolus , Sanatephorus ( Thana )
tephorus ), Rhizoctonia ( Rhizocto )
nia ), Coprinus ( Psatyrella ), Polyporus ( Polyp )
orus), Pikunoporasu (Pycnoporus),
Phlevia , Hygrophorosis ( Hy
grophoropsis), Agaricus (Agari
cus), Basuserumu (Vascellum), Kurushiburumu (Crucibulum), Supororumiera (S
porormiella), Pureu Lotus (Pleu
rotus ), Grifora ( Grifora ), Ganoderma ( Ganoderma ), Rangetes ( Lenzi )
tes ), Fanerochaet ( Phanerochaet
e ), Ceripolyopsis ( Ceriporiopsi)
s), Rigidoporasu (Rigidoporus), Fomitera (Fomitella), Torakideruma (Tra
chyderma ), Aspergillus ( Aspergi
lls ), Alternaria ( Alternari )
a), Botrytis (Botrytis), Nectria (Nectria), Myrothecium (Myrothec
ium ), Ceratospha area
eria), Sutakiridiumu (Stachylidi
um ) (Japanese Patent Application Laid-Open No. 10-174583), Stilbella (Japanese Patent Application Laid-Open No. 10-17458).
3), Sagenomera (Sagenomella) (JP-A-10-174583), Pyricularia (Pyricu
laria ), Myselioph tiger
ora ) or Citalidinium ( Schytalidi )
um ), Heterobasidium
ium), Fiforoma (Hypholoma), Reputoporasu (Leptoporus), Panas (Panu
s ), Stereum ( Stereum ), Roselinia ( R
osellinia), Fomitopushisu (Fomito
PSIS), it may include strains that are classified in each genus Foriota (pholiota) and Acremonium (Acremonium). In particular, the laccase-producing bacteria, Trametes Virosa (Trametes vi
llosa ) (Yaver, DS, et al .: Gene, 1).
81, 95-102 (1996); Yaver, D .;
S. Et al .: Appl. Environ. Microbio
l. , 62, 834-841 (1996)), Myseliophthora thermophila
thermophila ) (Randy, M. Ber
ka et al .: Abstracts of Papers A
merican Chemical Society
209 (1-2): BIOT 196, (199
5)), Citaridium Thermo Film ( Scyta
lidium thermophilum ) (Rand
y, M. Berka et al .: Abstracts of P
apers American Chemical S
society 209 (1-2): BIOT 196.
(1995)), Rhizoctonia solani ( Rhizoc
tonia solani ) (Wahleithne
r, J. A. Et al: Curr. Genet. 29,395
~ 403 (1996)), Myrothecium verrucaria
(WO97 / 00948), Myrothecium roridum (WO
97/00948), Coprinusc
inereus ) (Schneider, P et al .: Enz
ime Microb. Technol. 25,50
2 to 508 (1999)), oyster mushroom ( Pycnop )
orus coccineus ) (Oda, Y. et al .: A
gric. Biol. Chem. , 55, 1393-1
395 (1991), Coriolus
versicolor) (Morohoshi, N.
Et al .: Mokuzai Gakkaishi, 33, 21.
8 to 225 (1987)), Arakawakaratake ( Cor
iolus hirsutus ) (Kojima, Y .;
Et al .: J. Biol. Chem. , 265, 15224 ~
15230 (1990)), Suehirotake ( Schiz
ophyllum commune ) (De Vrie
s, O.S. M. H. Et al .: J. Gen. Microbio
l. , 132, 2817 to 2826 (1986)), oyster mushroom ( Pleurotus ostreatus )
(Girdina, P. et al .: Eur. J. Bioch.
em. 235, 508 to 515 (1996)), Betula mushroom ( Fomitella flaxinea ), Pyricularia oryzae ( Pyricularia o)
ryzae), Botrytis cinerea (Botryti
s cinerea ) (Zouari, N. et al .: App
l. Biochem. Biotechnol. , 15,
213-225 (1987)), Ebitake ( Trach )
yderma tsunodae ), sulmethaque ( Ri
gidoporus zonalis), Aspergillus awamori (Aspergillus awamor
i), Aspergillus two radiodurans (Aspergil
lus nidulans ), Aspergillus niger ( Aspergillus niger ), Aspergillus oryzae ( Aspergillus oryza)
e ), Aspergillus Tereus ( Aspergill
us terreus ), Aspergillus soyae ( A
spergillus sojae ), Acremonium murorum
(Gouka, R. J. et al .: Appl. Enviro.
n. Microbiol. 67, 2610-2616
(2001)) or variants thereof. Of these, Trametes sp. Laccase group derived from Ha1 strain (Patent Nos. 2984552 and 2984)
No. 584) is particularly preferable.

Any substance can be used in the method of the present invention so long as it has the laccase activity as described above.
Furthermore, plant-derived laccase may be used. For example, a laccase derived from Rhus vernicifera . A laccase-like enzyme with similar catalytic activity to laccase can be used. Such enzymes include, for example, mushrooms ( Agaric
Catechol oxidase (EC 1.10.3.1) from Us bisporus, Myrothecium verrucari
a ), Coprinus m
icaceus ) (U.S. Pat. No. 4,677,062), Pholiota nameko
Examples include bilirubin oxidase (EC1.3.3.5) derived from (US Pat. No. 4,677,062) and the like, and ceruloplasmin (EC1.16.3.1) derived from animals. Also, proteins with catalytic activity similar to laccase can be used. For example, snail ( Heli
x pomatia ), horseshoe crab ( Limulus )
polyphemus ) or spiny lobster ( Panuli )
hemocyanin derived from Rus interruptus ). The laccase or laccase-like enzyme or protein can be either one prepared from an organism (preferably a microorganism) isolated from nature and one produced from a genetically modified strain in which a gene has been cloned and expressed (Ong. , E. et al .: Gene, 1
96, 113-119 (1997); Mikuni,
J. Et al .: FEMS Microbiol. Lett. ，
155, 79-84 (1997); Hatamoto,
O. Et al: Biosci. Biotechnol. Bio
chem. , 63, 58-64 (1999)). Variants that may occur in genetic recombination or other manipulations are also
It can be used as long as it exhibits laccase activity. These bacteria may be used alone or in combination of two or more for the preparation of the enzyme.

Therefore, the "laccase-containing substance" used in the method of the present invention may be any substance containing the laccase or laccase-like enzyme or protein as described above and exhibiting laccase activity.
The laccase-containing material in the present invention includes a culture prepared from the above-mentioned microorganism (culture obtained by removing the microorganism by centrifugation), or a treated product thereof (for example, a partially purified or completely purified preparation of the culture). Can be used.

In the present invention, the microorganism which can be used for preparing the laccase-containing material (hereinafter, abbreviated as producing bacterium) can be cultivated in either liquid culture or solid culture. The growth medium for the production bacterium may be any medium used for culturing ordinary microorganisms, particularly fungi, and liquid medium or solid medium is used. Any carbon source may be used as long as it can be assimilated by the producing bacterium, such as sucrose, lactose, fructose, glucose,
Sorbitol, sugars such as starch, molasses, wheat bran, wood flour and the like can be used. As the nitrogen source, in addition to organic nitrogen sources such as peptone, yeast extract, malt extract, meat extract, soybean decomposition product, urea, amino acid, casamino acid, etc., inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can also be used. Inorganic salts such as phosphates, potassium, sodium, calcium, magnesium, manganese, cobalt, iron, copper, zinc, vitamins, nucleic acid-related compounds and the like can be added as necessary. These medium components may have a concentration that does not inhibit laccase production of the producing bacterium, the carbon source is 0.1 to 20% by weight, preferably 1 to 10% by weight, and the nitrogen source is 0.01 to 5% by weight. , Preferably 0.1 to 2% by weight.

Cultivation may be carried out by aeration culture or shaking culture in liquid culture, and static culture in solid culture, in accordance with ordinary mesophilic culturing. PH of culture solution
Is 3 to 9, preferably 6 to 8. The culture temperature is 10 to 50 ° C, preferably 20 to 40 ° C. The duration of culture is a period in which the desired laccase is sufficiently produced. Depending on the type of microorganism, it is usually 1-10
It is about 3 to 5 days, preferably about 3 to 5 days.

For the purification of the enzyme from the culture, known purification methods such as dialysis, salting out, various chromatographies, isoelectric focusing and gel filtration can be used alone or in combination.

The laccase-containing material used in the method of the present invention can be produced by preparation from an organism and genetic recombination as described above, but if it is an industrially mass-produced commercial laccase-containing material. Wise, from the perspective of cost reduction. Commercially available industrial laccase formulations are currently available from Trametes sp. Ha1 stock (Hatanaka Nakatani,
Masashi Shimizu: Journal of Japan Society for Agricultural Chemistry, Vol. 68, No. 03, p. 460 (1
Laccase [trade name: Laccase Daiwa (manufactured by Daiwa Kasei Co., Ltd.)] produced by 994)) is the only example in Japan.

(Laccase mediator (Lacc
In the present invention, a laccase mediator that can be used in combination with laccase refers to a substance having a property of enhancing and transmitting the catalytic reaction of laccase. The laccase mediator used in the present invention comprises (i) a low molecular weight (molecular weight 10
00 or less), (ii) moderate (0.4 to 1.2 V or less)
A substrate for laccase (iv) that has a redox potential, (iii) forms a stable radical,
(V) It may have properties such as no or little damage to laccase activity. Several kinds of laccase mediators are known, such as aromatic compounds and heterocyclic compounds (eg, N-hydroxyacetanilide, violuric acid, etc.). Suitable laccase mediators are desirable for the degradation of organic contaminants. Preferably, it is 1-hydroxybenzotriazole (1-hydroxybenzotriazole; hereinafter abbreviated as HBT).

The laccase mediator-containing substance used in the present invention may be in any form as long as it contains the laccase mediator, and preferably, the laccase mediator can be added after being dissolved in an organic solvent. Examples of such an organic solvent include hydrocarbons such as isooctane, octane and decane.
More preferably, the laccase mediator is soluble in the same or miscible organic solvent used in the method of the invention. For example, if the organic contaminant is solubilized with isooctane, preferably a solution of the laccase mediator in isooctane is used.

(Surfactant) The surfactant used in the present invention refers to any compound which forms a reverse micelle in an organic solvent and stabilizes the laccase-containing substance. Such surfactants may also include dyes and macromolecules that form reverse micelles and stabilize laccase-containing substances in organic solvents depending on the decomposition treatment conditions. Preferably, the surfactant is soluble in both the nonpolar solvent and the polar solvent. More preferably, di-2-ethylhexyl sodium sulfosuccinate (Di-2-ethylhexyl so)
trade name: A
erosol OT; hereinafter abbreviated as AOT) or cetyltrimethylammonium bromide (Cetyltr)
imethylammonium bromide; also known as: Hexadecyltrimethylammon
ium bromide; Trade name: Cetavlon;
Hereinafter, it is abbreviated as CTAB). Particularly preferably, AO
T. The reason why AOT is particularly preferred is that (i) the effect on the enzyme is small, (ii) the enzyme is less susceptible to denaturation, and (iii) the substrate moves near the interface. With regard to the properties of the reverse micelle formed thereby, (i) the stability as a micelle is good, (i)
i) Another reason is that the micelles are likely to be uniform because they are likely to be fused / separated within the micelles.

(Organic solvent) Organic pollutants are generally hardly soluble or insoluble in water. Therefore, in decomposing an organic pollutant using the method of the present invention, first, an organic solvent capable of dissolving the organic pollutant is added to waste water or a pollutant that may contain the organic pollutant, and then the organic pollutant is decomposed. The substance needs to be solubilized. The organic solvent used in the present invention is a water-insoluble or sparingly soluble organic solvent. Such an organic solvent may have properties such as (i) little volatility at room temperature and normal pressure, (ii) no toxicity, (iii) easy recovery. Examples of such an organic solvent include hydrocarbons such as isooctane, octane, and decane. Particularly preferred is isooctane.

(Organic Pollutant) In the present invention, the organic pollutant to be decomposed is not particularly limited. Using the method of the present invention, any compound that can be decomposed in an aqueous solution by a laccase-containing material or a combination of a laccase-containing material and a laccase / mediator-containing material has an increased dissolved oxygen content in an organic solvent as compared with water. And, due to the increased solubility of organic pollutants, are (efficiently) decomposed in organic solvents. Even organic compounds that are difficult to decompose in an aqueous solution due to their insolubility or poor solubility in water can be decomposed by acting an enzyme in an organic solvent. The compound that can be decomposed using the method of the present invention is a compound that has been conventionally recognized as a hardly biodegradable organic pollutant, for example, an organic halogen compound (chlorophenols (Roy).
-Arcand, L .; Et al: Enzyme Micro
b. Technol. , 13, 194-203 (199
1)), endocrine disrupting chemicals (Takaaki Tanaka, Masayuki Taniguchi et al .:
Proceedings of the 33rd Autumn Meeting of the Chemical Engineering Society of Japan (September 2000)
)), Azo compounds (Abadulla, E. et al .: Ap
pl. Environ. Microbiol. , 66,
3357-3362 (2000)), polycyclic aromatic hydrocarbons (Johannes, C. et al .: Appl. Envir.
on. Microbiol. , 66, 524-528
(2000)) and the like. Particularly preferred in such a group of aromatic compounds are o -chlorophenol, m -chlorophenol, p -chlorophenol,
2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol and bisphenol A. In addition, derivatives or analogs of the above compounds are also suitable compounds to be decomposed. Examples of such compounds to be decomposed include PCB and dioxin. In addition, pollutants decomposed by laccase in aqueous solution (Takaaki Tanaka, Masayuki Taniguchi et al .: Proceedings of the 33rd Autumn Meeting of the Chemical Engineering Society of Japan (September 2000)
Moon)) can also be suitably decomposed using the method of the invention.
Such compounds to be decomposed are, for example, octylphenol, nonylphenol, ethinylestradiol and the like.

As the organic halogen compound, chlorophenol, polychlorophenol, dioxin, PC
B, DDT, HCB, TCE, PBBE and the like are included, but are not limited thereto.

Examples of endocrine disrupting chemicals (environmental hormones) include, but are not limited to, alkylphenols, bisphenol A, phthalates, DES, and organic tin compounds.

(Method for decomposing organic pollutants) When decomposing an organic pollutant using the method of the present invention, a hydrophilic waste liquid containing the organic pollutant or one contaminated with the pollutant (for example, soil) , Container, equipment, etc.), the contaminant is preferably solubilized with the above-mentioned organic solvent and extracted. Thus obtained organic pollutant solubilized liquid, by using the laccase-containing material entrapped in reverse micelles, or by the combined use of the laccase-containing material and laccase mediators entrapped in reverse micelles, Dissolve pollutants. Details of the method for decomposing organic pollutants are as follows.

A surfactant was added to 1 part (volume basis) of a laccase solution obtained by dissolving the above-mentioned laccase-containing material in a buffer solution (eg, phosphate buffer solution, borate buffer solution, etc.) or water. Dissolved water-insoluble organic solvent 5
Mix at a rate of ~ 30 parts and form reverse micelles in a suitable manner. Examples of the organic solvent used for dissolving the surfactant include the organic solvents described above (for example, isooctane, octane, decane, etc.). This organic solvent may be the same as the organic solvent used to solubilize the organic contaminants described above. In order to form reverse micelles, any emulsification method known to those skilled in the art may be used, and examples of such a method include a stirring method, a rubbing method, and a combination method of both. Specifically, a stirring device (homogenizer, homomixer,
Reverse micelles may be formed by a magnetic stirrer, a mechanical stirrer, etc.) and, if necessary, ultrasonic irradiation. As a method for forming reverse micelles, a microinjection method can also be mentioned. The microinjection method is a laboratory-scale method in which an enzyme solution dissolved in a buffer solution is gradually added by injection or a microsyringe into an organic solvent (containing a surfactant and contaminants) and then a stirrer is used. This is a method of forming a microemulsion by stirring (100 rpm / min). In such a procedure, the surfactant directs lipophilic groups to the outer organic solvent and hydrophilic groups to the inner aqueous phase,
The inclusion of laccase inclusions in the inner aqueous phase. In order to maintain the stability of the reverse micelle, a salt such as Glauber's salt or a polymer compound such as dextrin may be added in a small amount or a small amount.

The reverse micelles encapsulating laccase contained in an organic solvent are allowed to act on the organic solution containing the organic contaminant prepared as described above or the organic contaminant itself. Here, "acting the reverse micelles ..." means that the enzyme (laccase) entrapped in the reverse micelles directly or indirectly exerts a catalytic action on the acted substance. The organic solution containing the organic pollutant or the organic pollutant itself may be added to the organic solvent in advance or after the reverse micelle formation. If necessary, laccase mediator may be added appropriately. The laccase mediator is added at different times depending on the purpose, but preferably before reverse micelle formation. The treatment conditions (eg, temperature, pH, etc.) can be such that the laccase-containing substance entrapped in the reverse micelle does not inactivate the laccase activity. The treatment temperature is preferably 40 to 70 ° C, more preferably 55 to 65 ° C. The treatment pH is under acidic to basic conditions, for example, pH 1 to 14, preferably pH 3 to 8,
More preferably, it is carried out at pH 5 to 7. Air exposure is sufficient for oxygen supply during the decomposition process,
It is preferable to carry out aeration and stirring if necessary. The decomposition process is performed in a bright place or a dark place. The treatment time is for the duration of the activity of the laccase entrapped in the reverse micelles or for the decomposition of the organic pollutants in the organic solvent, preferably about 1 to 24 hours.

Decomposition of organic pollutants is carried out by HPLC, GC-
It can be measured by measuring the decrease in the substrate by a known method such as MS.

Since the above-described decomposition treatment method treats highly hydrophobic organic pollutants as decomposition targets, the laccase-containing material contained in the reverse micelle in the form of an aqueous enzyme solution is
Even if the target pollutant dissolved in the water-insoluble organic solvent has a high concentration, it is obvious that it is not affected by the concentration inhibition.

Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to this range.

[0046]

Example ((Example 1) Comparison of laccase activity in isooctane in various reaction systems) Laccase Daiwa Y120 [manufactured by Daiwa Kasei Co., Ltd .; 120,000 POU /
g] is dissolved in a phosphate buffer (pH 6.0) to obtain a laccase solution. Next, a reverse micelle containing laccase was formed from the laccase solution and the isooctane solution containing AOT and o -chlorophenol by a microinjection method, and this was designated as AOT reverse micelle I system. Also, A
CTAB which replaced AOT of reverse micelle I system with CTAB
Both the reverse micelle system and the system in which lyophilized laccase was directly added to surfactant-free isooctane without being dissolved in a buffer (hereinafter referred to as lyophilized enzyme) were used as comparison targets. For these three reaction systems in total, 40 ° C, 10
The enzymatic reaction was carried out under the condition of 0 rpm warm shaking, and HPLC analysis was carried out for the change with time of the decomposition rate of o -chlorophenol. The HPLC equipment used was a Shimadzu product. The HPLC analysis program holds 70:30 (1% phosphate buffer: acetonitrile) for 5 minutes,
It was made to reach 0:90 in 5 minutes, held in this state for 10 minutes, then returned to 70:30 and held for 10 minutes. As the column, Inertosyl ODS-3 was used. The detection wavelength was 280 nm. The final concentration of each compound at the start of the reaction in each reaction system is as shown in Table 1.

[0047]

[Table 1] FIG. 1 shows the change with time of the decomposition rate of o -chlorophenol in each reaction system up to 24 hours after the start of the reaction. As can be seen from FIG. 1, the degradation rate at 24 hours after the start of the reaction was 80% or more in the AOT reverse micelle I system, about 20% or less in the CTAB reverse micelle system, and 0% in the freeze-dried enzyme.

(Example 2) Effect of final concentration of AOT in AOT reverse micelle I system In the AOT reverse micelle I system of Example 1, the final concentration of AOT was 50 at 40 ° C. under the reaction condition of 100 rpm. , 100, 150, 200,
When changed to 250 mM, each o -chlorophenol decomposition rate 6 hours after the start of the reaction was calculated by HPLC analysis. As a result, as shown in FIG. 2, the highest decomposition rate was obtained at the final concentration of 100 mM.

(Example 3) Influence of pH in AOT reverse micelle I system In the AOT reverse micelle I system of Example 1, the reaction solution was adjusted to pH 3.0 under the reaction conditions of 40 ° C. and 100 rpm. 4.0, 5.0, 6.0, 7.
It was changed to 0 and 8.0, each o 6 hours after the start of the reaction.
-Chlorophenol decomposition rate was calculated by HPLC analysis. As a result, as shown in FIG. 3, the highest decomposition rate was obtained at pH 6.0.

(Example 4) Effect of Wo value in AOT reverse micelle I system In the AOT reverse micelle I system of Example 1, the Wo value of the reaction solution was 10 under the reaction conditions of 40 ° C. and 100 rpm. Variously changed in the range of up to 50
Each o -chlorophenol decomposition rate at the o value 6 hours after the start of the reaction was calculated by HPLC analysis. The result is shown in FIG.

As can be seen from FIG. 4, Wo = 10-35
In the case of Wo = 40 and 45, the decomposition rate of o -chlorophenol increased and the decomposition rate of o -chlorophenol was almost the same. At Wo = 50, the water content became too high and no reverse micelle was formed. From the above, when Wo = 40,
It was concluded that laccase is most stably incorporated in the state.

(Example 5) Influence of reaction temperature in AOT reverse micelle I system In the AOT reverse micelle I system of Example 1, under the reaction conditions of 40 ° C. and 100 rpm heat and shaking.
The reaction temperature of the reaction solution is 40, 45, 50, 55, 60, 6
The decomposition rate of each o -chlorophenol after 1 hour from the start of the reaction was calculated by HPLC analysis while changing to 5, 70, and 75 ° C. As a result, the highest decomposition rate was obtained at 60 ° C. as shown in FIG.

(Example 6) Effect of Enzyme Concentration in AOT Reverse Micelle I System In AOT reverse micelle I system of Example 1, under the reaction conditions of 40 ° C. and 100 rpm heat and shaking.
The enzyme final concentration of the reaction solution is variously changed in the range of 0 to 15 μM,
Each o -chlorophenol decomposition rate 1 hour after the start of the reaction was calculated by HPLC analysis. As a result, as shown in FIG. 6, when the concentration was 9 μM or more, a decomposition rate of 80% or more was obtained.

(Example 7) Decomposition of chlorophenols in the AOT reverse micelle I system In the AOT reverse micelle I system of Example 1, various substrates of the reaction solution were used under the reaction conditions of 40 ° C. and 100 rpm heat and shake. Instead, the decomposition rate of each substrate 3 hours after the start of the reaction was calculated by HPLC analysis. As a result, the decomposition rates shown in Table 2 were obtained.

[0055]

[Table 2] ((Example 8) Identification of Decomposition Products Accompanying p -Chlorophenol Decomposition in AOT Reverse Micelle I System) In the AOT reverse micelle I system of Example 1, the reaction was carried out under the reaction conditions of 40 ° C. and 100 rpm incubation. the substrate for liquid p - decomposition products when changing the chlorophenol, p after the start of the reaction for 3 hours - the decomposition products from chlorophenol was mass spectrometry were trimethylsilylated. FIG. 7 shows the mass spectrum data of the standard product, and FIG. 8 shows the mass spectrum data of the decomposition product. From the comparison between FIG. 7 and FIG. 8, the decomposition product was found to be catechol. That is, laccase was able to dechlorinate p -chlorophenol in the AOT reverse micelle I system.

((Example 9-1) Decomposition of organic pollutants in AOT reverse micelle II system in the presence of laccase / mediator) Laccase Daiwa Y120 (manufactured by Daiwa Kasei Co., Ltd .; 120,000 POU / g) was added to phosphorus. It is dissolved in an acid buffer (pH 3.0) to obtain a laccase solution. Then, a microinjection method was used to form a reverse micelle containing laccase from a laccase solution and an isooctane solution containing 1-hydroxybenzotriazole (HBT), AOT and an organic pollutant.
・ HBT coexistence system was adopted. This AOT reverse micelle II HB
With respect to the T coexisting system, an enzymatic reaction was carried out under the condition of 45 ° C. and 100 rpm while being kept warm, and the decomposition rate of organic pollutants 2 hours after the reaction was started was calculated by HPLC analysis. As organic pollutants to be decomposed, bisphenol A, o-chlorophenol, 2,4-dichlorophenol, and 2,
4,6-Trichlorophenol was used. Table 3 shows the final concentration of each compound at the start of the reaction of each reaction system, and Table 4 shows the data of the decomposition rate of each organic pollutant.

(Example 9-2) Decomposition of organic pollutants in AOT reverse micelle II system Laccase Daiwa Y12
0 [Daiwa Kasei Co., Ltd .; 120,000 POU / g] is dissolved in a phosphate buffer (pH 3.0) to obtain a laccase solution. Next, by microinjection method, laccase solution and AO
A reverse micelle entrapping laccase was formed from an isooctane solution containing T and an organic contaminant, and this was designated as AOT reverse micelle II system. This AOT reverse micelle II
The system was subjected to an enzymatic reaction under the conditions of 45 ° C. and 100 rpm while being shaken, and the decomposition rate of organic pollutants 2 hours after the start of the reaction was calculated by HPLC analysis. In addition, as organic pollutants to be decomposed, bisphenol A, o -chlorophenol, 2,4-dichlorophenol, and 2,4,6-
Trichlorophenol was used. Table 3 shows the final concentration of each compound at the start of the reaction of each reaction system, and Table 4 also shows the result of the decomposition rate of each organic pollutant.

[0058]

[Table 3]

[0059]

[Table 4] According to the present invention, for example, by using the AOT reverse micelle II system, in the presence or absence of HBT, which is a laccase mediator, 2,4-dichlorophenol is 70.1%, It shows a decomposition rate of 0.3%. Further, by using the AOT reverse micelle I system, 2,4-dichlorophenol can be decomposed by 91% 3 hours after the reaction start even in the absence of HBT. In addition, p -chlorophenol is dechlorinated from the AOT reverse micelle I system.

[0060]

INDUSTRIAL APPLICABILITY The present invention provides a method for easily and quickly decomposing an organic contaminant existing in an organic solvent by using laccase or by using laccase and a laccase mediator in combination.
INDUSTRIAL APPLICABILITY The present invention provides an energy-saving and clean decomposition method capable of simultaneously and efficiently decomposing organic pollutants as compared with known decomposition methods.

[Brief description of drawings]

FIG. 1 is a graph showing the time course of o -chlorophenol decomposition reaction when using the AOT reverse micelle I system, the CTAB reverse micelle system, and the freeze-dried enzyme system.

FIG. 2 is a graph showing AOT concentration dependence of o -chlorophenol decomposition reaction in the AOT reverse micelle I system.

FIG. 3 is a graph showing the pH dependence of the o -chlorophenol decomposition reaction in the AOT reverse micelle I system.

FIG. 4 is a graph showing the Wo dependence of the o -chlorophenol decomposition reaction in the AOT reverse micelle I system.

FIG. 5 is a graph showing the reaction temperature dependence of o -chlorophenol decomposition reaction in the AOT reverse micelle I system.

FIG. 6 is a graph showing the enzyme concentration dependence of the o -chlorophenol decomposition reaction in the AOT reverse micelle I system.

FIG. 7 is mass spectrum data of a standard product (a trimethylsilylated derivative of catechol) in Example 8.

FIG. 8 is mass spectrum data of a trimethylsilylated derivative of a decomposition product of p -chlorophenol in Example 8.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12R 1:66) C12R 1: 685 (C12N 9/02 1: 785 C12R 1: 665) C12R 1:69 ( (C12N 9/02 C12R 1: 685) (C12N 9/02 C12R 1: 785) (C12N 9/02 C12R 1:69) (72) Inventor Shinya Okazaki 1-1 Ariakidai Higashi, Ichihara City, Chiba Mitsui Nagato Dormitory E2 -303 (72) Inventor Junji Dozoe 18-504 F-term (Reference) 4-B050 CC08 CC10 DD03 DD05 HH01 KK03 KK06 KK11 LL10 4B065 AA60X AA62Y AA63X AA66X AA71X AC14 BB06 CA28 CA56

Claims (17)

[Claims] 1. A method for expressing laccase activity in an organic solvent, which comprises the step of incorporating a laccase-containing substance into reverse micelles using a surfactant involved in reverse micelle formation. 2. The method according to claim 1, characterized in that the method comprises using the reverse micelle in the presence of a laccase mediator-containing substance to enhance and transfer laccase activity in an organic solvent. 3. The organic solvent contains an organic pollutant, and the organic pollutant is decomposed.
Or the method described in 2. 4. The method according to any one of claims 1 to 3, wherein the surfactant is sodium di-2-ethylhexyl sulfosuccinate or cetyltrimethylammonium bromide. 5. The organic solvent according to claim 1, which is insoluble or sparingly soluble in water.
The method described in the section. 6. The method according to claim 1, wherein the organic solvent is isooctane. 7. The method according to claim 2, wherein the laccase mediator-containing material is a 1-hydroxybenzotriazole-containing material. 8. The organic pollutant of claim 3, wherein the organic pollutant comprises a chemical selected from the group consisting of organic halogen compounds, endocrine disrupting chemicals, azo compounds, and polycyclic aromatic hydrocarbons. The method according to any one of items. 9. The organic pollutant is o -chlorophenol, m -chlorophenol, p -chlorophenol,
9. A chemical substance selected from the group consisting of 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, and bisphenol A, according to any one of claims 3 to 8. The method according to item 1. 10. The laccase-containing material is a culture of a laccase-producing bacterium or a processed product thereof.
The method according to any one of 1. 11. The method according to claim 10, wherein the laccase-producing bacterium is genus Tramet.
es sp. ) The above method, which is Ha1 strain. 12. The laccase-containing material entrapped in a reverse micelle with sodium di-2-ethylhexyl sulfosuccinate has the following decomposition characteristics with respect to o -chlorophenol dissolved in isooctane: (1) Stabilizing action The method according to any one of claims 3 to 11, which has a pH range: pH 3.0 to 8.0; (2) a stable action temperature range: 40 ° C to 75 ° C. 13. The laccase-producing bacterium is selected from the group consisting of a strain belonging to the subphylum Zygomycete, a strain belonging to the subphylum Ascomycete, a strain belonging to the subphylum Basidiomycete, and a strain belonging to the subphylum Incomplete. The method according to claim 10. 14. The method according to claim 13, wherein the laccase-producing bacterium is Mucor , Neurospora , Podspora ( P ).
odospora ), Colivia ( Collybia ),
Fomesu (Fomes), Lentinus (Lentinu
s ), Schizophyllum ,
Trametes , Coriolas ( Cor
iolus), Sanateforasu (Thanatepho
rus ), Rhizoctonia ( Rhizoctonia ),
Coprinus , Psachillera ( Psa )
Tyrrella ), Polyporus ( Polyporu )
s), Pikunoporasu (Pycnoporus), Furebia (Phlebia), Higurohoropushisu (Hygro
phoropsis), Agaricus (Agaricu
s), Basuserumu (Vascellum), Kurushiburumu (Crucibulum), Supororumiera (Spo
rormiella), Pureu Lotus (Pleuro
tus ), Grifora ( Grifora ), Ganoderma ( Ganoderma ), Lenzite ( Lenzite )
s ), Phanerochaet
e ), Ceripolyopsis ( Ceriporiopsi)
s), Rigidoporasu (Rigidoporus), Fomitera (Fomitella), Torakideruma (Tra
chyderma ), Aspergillus ( Aspergi
lls ), Alternaria ( Alternari )
a), Botrytis (Botrytis), Nectria (Nectria), Myrothecium (Myrothec
ium ), Ceratospha area
eria), Sutakiridiumu (Stachylidi
um), Sutirubera (Stilbella), Sagenomera (Sagenomella), Pyricularia (P
yricularia ), Miseriophora ( Mycel )
iophtora), Shitarijiumu (Schytali
dium ), Heterobasium ( Heterobas
idium), Fiforoma (Hypholoma),
Reputoporasu (Leptoporus), Panas (Pa
nus), Sutereumu (Stereum), Roserinia (Rosellinia), Fomitopushisu (Fomi
toposis ), foliota ( Pholiota ) and Acremonium ( Acremonium ). 15. The method according to claim 14, wherein the laccase-producing bacterium is Trametes virsosa ( Tr.
ametes villosa), Miseri off Torah
Thermophila ( Myceliophtora ther
mofila ), Citalidium thermofilm ( Scytalidium thermophilu
m ), Rhizoctonia solani ( Rhizoctonia )
solani ), Milocesium vell carrier ( M
yrothecium verrucaria), Myrothecium Roridamu (Myrothecium ror
idum ), Coprinus cine
reus ), oyster mushroom ( Pycnoporus co )
ccineus ), kawaratake ( Coriolusve )
rsicolor), Coriolus hirsutus (Coriol
us hirsutus ), Suehirotake ( Schiz )
ophyllum commune ), oyster mushroom ( Pl
eurotus ostreatus ), beetle mushroom ( Fomitella flaxinea ), Pyricularia oryzae ( Pyricularia oryz)
ae ), Botrytis cinerea ( Botrytis c)
inerea), Ebitake (Trachyderma
tsunodae), Surumetake (Rigidopor
us zonalis ), Aspergillus awamori ( Aspergillus awamori ), Aspergillus nidulans ( Aspergillus n)
Idulans ), Aspergillus niger ( Aspe
rgillus niger), Aspergillus oryzae (Aspergillus oryzae), Aspergillus terreus (Aspergillus te
rreus ), Aspergillus soyae ( Asperg
illussojae ), Acremonium murorum, and variants thereof. 16. A method for degrading organic pollutants, comprising the steps of: mixing a laccase-containing material with an organic solvent containing a surfactant to form reverse micelles containing laccase. And the step of allowing the reverse micelle to act. 17. The method of claim 16, further comprising the step of mixing the laccase mediator containing material.