KR101185823B1 - Transformant expressing two kinds of ligninases, method for preparing the transformant and method for degrading endocrine disruptor by using the transformant - Google Patents

Transformant expressing two kinds of ligninases, method for preparing the transformant and method for degrading endocrine disruptor by using the transformant Download PDF

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KR101185823B1
KR101185823B1 KR1020090132795A KR20090132795A KR101185823B1 KR 101185823 B1 KR101185823 B1 KR 101185823B1 KR 1020090132795 A KR1020090132795 A KR 1020090132795A KR 20090132795 A KR20090132795 A KR 20090132795A KR 101185823 B1 KR101185823 B1 KR 101185823B1
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laccase
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이성숙
유선화
최형태
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Abstract

본 발명은 2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용한 환경호르몬 분해방법에 관한 것으로, 보다 상세하게는 락카아제 유전자와 망간 퍼옥시다아제 유전자를 동시에 발현시키는 것을 특징으로 하는 재조합벡터 pMnP-gLac 및 그의 제조방법; 상기 재조합 벡터 pMnP-gLac로 형질전환된 것을 특징으로 하는 기계충버섯 또는 아교버섯의 형질전환체 및 그의 제조방법; 및 상기 형질전환체를 이용하여 리그닌 또는 환경호르몬을 분해하는 방법에 관한 것이다.The present invention relates to two kinds of lignin degrading enzyme expression transformant, a method for preparing the same and an environmental hormone decomposition method using the same, and more particularly, a recombinant vector characterized by simultaneously expressing a laccase and a manganese peroxidase gene. pMnP-gLac and its preparation method; A transformant of a mushroom or a gluco mushroom, characterized in that transformed with the recombinant vector pMnP-gLac, and a method for producing the same; And it relates to a method for degrading lignin or environmental hormone using the transformant.

최근 보편화된 일회용 용기 등의 사용으로 인해 환경호르몬이 인간의 체내에 축적되어 체내 호르몬의 정상적인 작용을 방해함으로써 불임(不姙), 기형아 출산 또는 암 등의 질병을 일으키는 문제점이 발생하고 있다. 이에 리그닌 분해효소를 가진 백색부후균이라 불리는 버섯이 환경호르몬 분해를 위해 사용되고 있다. 그러나 종래 백색부후균의 형질전환체는 락카아제 또는 망간 퍼옥시다아제 중 하나의 유전자만을 삽입한 형질전환체로서 생물학적 분해능을 실질적으로 미흡한 단점이 있었다. Recently, environmental hormones accumulate in the human body due to the use of general-purpose disposable containers, which interferes with the normal function of hormones in the body, causing problems such as infertility, birth defects, or diseases such as cancer. Thus, mushrooms called white rot fungi with lignin degrading enzymes are used for the degradation of environmental hormones. However, the transformants of the conventional white fungi have a disadvantage in that the transformants inserting only one gene of laccase or manganese peroxidase have substantially insufficient biodegradability.

이에 본원 발명은 비스페놀 A와 벤질부틸프탈레이트 등과 같은 환경호르몬 분해능을 극대화 시키기 위한 락카아제 및 망간 퍼옥시다아제 유전자가 동시에 발현하는 재조합 벡터를 도입한 형질전환체를 제공한다.Accordingly, the present invention provides a transformant that introduces a recombinant vector which simultaneously expresses laccase and manganese peroxidase genes to maximize environmental hormone resolution, such as bisphenol A and benzylbutyl phthalate.

형질전환체, 백색부후균, 리그닌, 락카아제, 망간 퍼옥시다아제, 겨울우산버섯, 아교버섯, 기계충버섯, 환경호르몬 Transformants, white rot fungi, lignin, laccase, manganese peroxidase, winter umbrella, glue mushroom, mechanical mushroom, environmental hormone

Description

2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용한 환경호르몬 분해방법{Transformant expressing two kinds of ligninases, method for preparing the transformant and method for degrading endocrine disruptor by using the transformant}Transformant expressing two kinds of ligninases, method for preparing the transformant and method for degrading endocrine disruptor by using the transformant}

본 발명은 본 발명은 2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용한 환경호르몬 분해방법에 관한 것이다. 보다 상세하게는 락카아제(laccase) 유전자와 망간 퍼옥시다아제(Mn peroxidase) 유전자를 동시에 발현시키는 것을 특징으로 하는 재조합벡터 pMnP-gLac 및 그의 제조방법; 상기 재조합 벡터 pMnP-gLac로 형질전환된 것을 특징으로 하는 기계충버섯 또는 아교버섯의 형질전환체 및 그의 제조방법; 및 상기 형질전환체를 이용하여 리그닌 또는 환경호르몬을 분해하는 방법에 관한 것이다.The present invention relates to two kinds of lignin degrading enzyme-expressing transformant, a method for preparing the same and an environmental hormone decomposition method using the same. More specifically, the recombinant vector pMnP-gLac and a method for producing the same, characterized by simultaneously expressing a laccase gene and a manganese peroxidase gene; A transformant of a mushroom or a gluco mushroom, characterized in that transformed with the recombinant vector pMnP-gLac, and a method for producing the same; And it relates to a method for degrading lignin or environmental hormone using the transformant.

환경호르몬은 우리가 편리한 생활을 위해 산업 활동을 함으로써 만들어지는 환경오염물질로, 이에 속하는 물질로는 컵라면 등 일회용 용기에 사용되는 스타일 렌(styrene), 플라스틱 제조시 가소제로 사용되는 프탈레이트류(phthalates), 음료수 캔의 내부 코팅제로 사용되는 비스페놀 A(bisphenol A), 세제 등에 포함되어 있는 노닐페놀(nonylphenol)과 옥틸페놀(octylphenol)등이 있다. 이러한 물질들은 물에 녹지 않는 소수성의 특징을 가지고 있어 적은 양으로도 생태계의 먹이사슬을 통하여 사람 또는 어류 등의 몸속에 축적되어 진짜 호르몬의 정상적인 작용을 방해하는 가짜 호르몬 역할을 하기 때문에 환경호르몬이라고 불리게 되었다. Environmental hormones are environmental pollutants produced by our industrial activities for convenient living. Styrenes used in disposable containers such as cup noodle and phthalates used as plasticizers in plastics are produced. ), Bisphenol A used as an inner coating agent of beverage cans, and nonylphenol and octylphenol included in detergents. These substances are hydrophobic, insoluble in water, and they are called environmental hormones because they accumulate in the body of humans or fish through the food chain in small amounts and act as fake hormones that interfere with the normal functioning of real hormones. It became.

상기 환경호르몬 피해의 대표적인 사례로서 여성 호르몬인 에스트로겐처럼 작용함으로써 남성이 여성화되는 것을 들 수 있다. 또한, 환경호르몬이 사람의 몸속에 쌓이게 되면 생식능력을 저하시켜 불임이 된다거나 기형 또는 암이나 아토피성 피부염 등의 병을 일으키게 된다.Representative examples of the environmental hormone damage is that the male is feminized by acting like the female hormone estrogen. In addition, when environmental hormones accumulate in a person's body, the fertility is reduced, resulting in infertility, or malformation or cancer or atopic dermatitis.

그런데 이렇게 인체에 나쁜 영향을 미치는 환경호르몬을 버섯으로 분해할 수 있다. 그러나 모든 버섯이 환경호르몬을 분해할 수 있는 것은 아니고 주로 목질 성분 중 리그닌을 분해해서 영양분을 섭취하는 백색부후균이라 불리는 버섯만이 환경호르몬을 분해할 수 있다. By the way, the environmental hormones that adversely affect the human body can be broken down into mushrooms. However, not all mushrooms can break down environmental hormones, and only mushrooms called white rot fungi, which break down lignin from wood components and consume nutrients, can break down environmental hormones.

백색부후균은 목질에 포함된 리그닌, 헤미셀룰로오스, 셀룰로오스를 모두 분해할 수 있으며, 상기 백색부후균이 견고한 구조의 나무를 부후시켜 분해함으로써 산림생태에서 영양소의 재순환에 중요한 역할을 담당한다. White fungus can decompose all lignin, hemicellulose, cellulose contained in the wood, and the white fungus plays an important role in the recycling of nutrients in forest ecology by decomposing and decomposing the wood of a solid structure.

한편 상기 리그닌 분해에는 대표적인 리그닌 분해효소인 락카아제(laccase), 리그닌 퍼옥시다아제(lignin peroxidase) 그리고 망간 퍼옥시다아제(Mn peroxidase)가 이용된다. On the other hand, the lignin decomposition is a typical lignin degrading enzyme laccase (laccase), lignin peroxidase (lignin peroxidase) and manganese peroxidase (Mn peroxidase) is used.

이러한 리그닌 분해효소들을 기질특이성이 낮아 리그닌과 구조가 유사한 환경호르몬도 분해할 수 있다, 즉, 버섯 가운데 기계충버섯, 아교버섯, 구름버섯, 조개껍질버섯 등 백색부후균은 리그닌을 분해하는 효소를 가지고 있으며 이 효소들은 리그닌과 구조가 유사한 환경호르몬도 분해할 수 있는 것이다.These lignin degrading enzymes have low substrate specificity, which can degrade environmental hormones similar in structure to lignin. In other words, white fungi, such as mechanical mushrooms, glue mushrooms, cloud mushrooms, shell mushrooms, etc. These enzymes can also degrade environmental hormones that are similar in structure to lignin.

지금까지 이러한 백색부후균의 형질전환은 몇몇 연구자들에 의해 시도되고 있다. 백색부후균 연구의 모델로 이용되고 있는 판막버섯은 제한효소를 이용한 형질전환 기법(Restriction enzyme mediated integration, REMI)을 통해 재조합 망간퍼옥시다아제 유전자를 도입하여 발현을 하였고(Ma B., et.. al., 2003 Curr Gent. 43(6):407-414), 아그로박테리움을 이용한 형질전환을 통해 외래에서 도입된 형광유전자와 항생물질유전자 등의 발현이 성공하였다(Sharma KK. et. al., 2006 Biotechnol. Appl Biochem. 43:181-186).To date, the transformation of these white fungi has been attempted by several researchers. Valve mushrooms, which are used as a model for the study of white fungus, were expressed by introducing recombinant manganese peroxidase gene through restriction enzyme mediated integration (REMI) (Ma B., et .. al. , 2003 Curr Gent. 43 (6): 407-414), and the expression of fluorescent genes and antibiotic genes introduced in foreign countries through the transformation with Agrobacterium was successful (Sharma KK. Et. Al., 2006 Biotechnol.Appl Biochem. 43: 181-186).

일본에서는 느타리버섯에서 제한효소를 이용한 형질전환법을 통해 자체 퍼옥시다아제 유전자를 재조합하여 도입한 후 붉은색 계통의 염료(Poly R-478)의 탈색능을 증가시켰다(Takahisa T. et. al., 2006 Applied microbiology and biotechnology 71(1) 114-120). In Japan, the red color dye (Poly R-478) increased the depigmentation ability after recombining its own peroxidase gene through transformation with restriction enzymes in oyster mushroom (Takahisa T. et. Al., 2006 Applied microbiology and biotechnology 71 (1) 114-120).

국내에서는 아교버섯과 기계충버섯을 제한효소를 이용한 형질전환법을 통해 포스피노트라이신 항생제에 대한 저항성을 갖는 형질전환체를 얻는데 성공하였다(김 외 2007 한국미생물학회지 43(2) 147-149). In Korea, we successfully obtained a transformant resistant to phosphinotricin antibiotics by transforming glucobacteria and machine mushrooms using restriction enzymes (Kim et al. 2007 (21) 147-149) .

본 발명에 관련된 종래기술은 한국특허등록 2003-0052251(백색부후균을 함유한 폐수처리용 고정화 담체, 그제조방법 및 이 담체를 이용한 폐수처리방법)로서, 백색부후균을 함유하는 폐수처리용 고정화 담체를 사용하면, 종래의 활성슬러지법에서 발생하는 슬러지 벌킹, 다량의 폐슬러지 발생, 폐수특성(pH, 온도, 독성물질 등)에 따른 충격부하 등의 문제 및 생물막공법의 운전중에 막에서 유출된 미생물의 침전성 저하로 인한 문제 등을 방지하면서 안정적으로 폐수를 처리할 수 있는 고농도 유기성폐수, 난분해성 폐수, 색도유발 폐수 처리 방법에 관한 것이다.The prior art related to the present invention is Korean Patent Registration 2003-0052251 (Immobilization carrier for wastewater treatment containing white fungus, manufacturing method thereof and wastewater treatment method using the carrier), Immobilization for wastewater treatment containing white fungus When the carrier is used, problems such as sludge bulking generated in the conventional activated sludge method, generation of a large amount of waste sludge, impact load according to the wastewater characteristics (pH, temperature, toxic substances, etc.), and leakage from the membrane during the operation of the biofilm process The present invention relates to a method for treating high concentration organic wastewater, hardly degradable wastewater and chromaticity-induced wastewater, which can stably treat wastewater while preventing problems due to deterioration of microorganisms.

또한 한국특허등록 10-0801983(환경호르몬 제거용 리포좀-앱타머 복합체 및 이를 이용한 환경호르몬 제거방법)로서, 핵산 앱타머를 리포좀 내에 위치시킨 리포좀-앱타머 구조체를 이용하여 용액 내에 존재하는 17베타-에스트라다이올을 특이적으로 제거하는 환경호르몬 제거용 방법에 관한 것이다. 그러나 이들 종래기술은 본 발명과 기술적구성이 다른 것이다.In addition, Korean Patent Registration 10-0801983 (enzyme removal method for environmental hormone liposome-aptamer complex and environmental hormone using the same), using a liposome-aptamer structure in which the nucleic acid aptamer is placed in liposome 17beta- The present invention relates to a method for removing environmental hormone that specifically removes estradiol. However, these prior art is different from the present invention in the technical configuration.

종래 백색부후균의 형질전환체는 락카아제(laccase) 또는 망간 퍼옥시다아제(Mn peroxidase) 하나의 유전자만을 삽입한 형질전환체로서, 리그닌 및 리그닌과 구조가 유사한 환경호르몬의 생물학적 분해능을 실질적으로 미흡한 단점이 있었다.Conventional white fungal transformants are laccases or manganese peroxidase transformants that insert only one gene, and have a disadvantage in that they substantially lack the biological resolution of an environmental hormone similar in structure to lignin and lignin. There was this.

이에 본 발명의 목적은 목재의 리그닌 및 리그닌과 구조가 유사한 환경호르몬의 생물학적 분해능을 극대화 시키기 위해 2종의 리그닌 분해효소 유전자가 동시에 발현하는 형질전환체를 제작하고 이를 이용하여 환경호르몬을 분해하고자 함에 있다.Accordingly, an object of the present invention is to prepare a transformant expressing two lignin degrading enzyme genes simultaneously to maximize the biological resolution of lignin and environmental hormones similar in structure to lignin and to degrade environmental hormones using the same. have.

상기의 과제를 해결하기 위해 본 발명에서는 환경호르몬을 분해하는 우수 균주 중 기계충버섯(Irpex lacteus)과 아교버섯(Phlebia tremellosa)을 선택하여, 리그닌 분해효소 중 락카아제 및 망간 퍼옥시다아제 유전자가 동시에 발현하는 재조합 벡터를 도입한 형질전환체 및 그의 제조방법을 제공한다.In order to solve the above problems, the present invention selects mechanical mushrooms (Irpex lacteus) and glue mushrooms (Phlebia tremellosa) among the excellent strains that degrade environmental hormones, expression of laccase and manganese peroxidase genes in lignin degrading enzymes simultaneously Provided is a transformant introducing a recombinant vector and a method for producing the same.

또한 본 발명에서는 상기 재조합 벡터를 도입한 형질전환체의 극대화된 분해능을 통한 비스페놀 A(bisphenol A)와 벤질부틸프탈레이트(benzylbutyl phthalate) 등의 환경호르몬 분해방법을 제공한다.The present invention also provides a method for degrading environmental hormones such as bisphenol A (benzylbutyl phthalate) and bisphenol A (maximum resolution) through the maximal resolution of the transformant introduced with the recombinant vector.

본 발명인 2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용하여 환경호르몬을 분해하는 방법을 통해, 목재의 리그닌 또는 리그닌과 구조가 유사한 비스페놀 A와 벤질부틸프탈레이트 등의 환경호르몬을 생물학적으로 분해함에 있어, 기계충버섯과 아교버섯을 선택하여 리그닌 분해효소 중 라카아제 및 망간 퍼옥시다아제 유전자가 동시에 발현하는 재조합 벡터를 도입한 형질전환체를 제공함으로써, 리그닌 또는 환경호르몬 분해능을 극대화시키는 시키는 유리한 효과가 있다.Through two lignin degrading enzyme-expressing transformants of the present invention, a method of preparing the same, and a method of decomposing environmental hormones using the same, biological hormones such as bisphenol A and benzyl butyl phthalate similar in structure to lignin or lignin of wood In the degradation, by selecting a mechanical mushroom and glue mushrooms to provide a transformant introducing a recombinant vector expressing the laccase and manganese peroxidase gene of the lignin degrading enzyme at the same time, to maximize the lignin or environmental hormone resolution It has a beneficial effect.

본 발명은 2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용하여 환경호르몬을 분해하는 방법에 관한 것에 관한 것이다.The present invention relates to two kinds of lignin degrading enzyme expression transformants, a method for preparing the same, and a method for degrading environmental hormones using the same.

보다 상세하게는 첫째, 락카아제(laccase) 유전자와 망간 퍼옥시다아제(Mn peroxidase) 유전자를 동시에 발현시키는 것을 특징으로 하는 재조합벡터 pMnP-gLac 및 그의 제조방법을 제공한다.More specifically, the present invention provides a recombinant vector pMnP-gLac and a method for producing the same, wherein the laccase gene and the manganese peroxidase gene are simultaneously expressed.

둘째, 상기 재조합 벡터 pMnP-gLac로 형질전환된 것을 특징으로 하는 기계충버섯(Irpex lacteus) 또는 아교버섯(Phlebia tremellosa)의 형질전환체 및 그의 제 조방법을 제공한다.Second, the present invention provides a transformant of Irpex lacteus or Phlebia tremellosa , and a method for producing the same , which are transformed with the recombinant vector pMnP-gLac.

셋째, 상기 재조합 벡터 pMnP-gLac로 형질전환된 것을 특징으로 하는 기계충버섯(Irpex lacteus) 또는 아교버섯(Phlebia tremellosa)의 형질전환체를 이용하여 리그닌 또는 환경호르몬을 분해하는 방법을 제공한다.Third, the present invention provides a method for degrading lignin or environmental hormone by using a transformant of Irpex lacteus or Phlebia tremellosa , which is transformed with the recombinant vector pMnP-gLac.

그리고 상기 락카아제 유전자와 망간 퍼옥시다아제 유전자를 동시에 발현시키는 재조합벡터 pMnP-gLac를 도입한 형질전환체의 제조방법은 제한효소 매개 삽입법(Restriction enzyme mediated integration)을 이용할 수 있다.In addition, a method for preparing a transformant into which the recombinant vector pMnP-gLac, which simultaneously expresses the laccase gene and the manganese peroxidase gene, may use restriction enzyme mediated integration.

삭제delete

바람직하게는 상기 제한효소 매개 삽입법은 벡터 DNA를 제한효 소(restriction enzyme)로 절단하는 단계; 상기 절단된 벡터 DNA를 STC 버퍼(buffer)에 현탁 되어 있는 300 ㎕의 원형질체에 넣는 단계; 상기 혼합된 벡터 DNA 및 원형질체를 40% 폴리에틸렌글리콜(polyethlene glycol) 용액을 섞어서 상온에서 20분 반응시키고, CKMM 액체배지에서 20~30℃의 온도로 15~17시간 배양하며, 채취하는 단계;로 구성될 수 있다.Preferably the restriction enzyme mediated insertion method comprises the steps of cleaving the vector DNA with a restriction enzyme (restriction enzyme); Placing the cleaved vector DNA in 300 μl of protoplasts suspended in an STC buffer; The mixed vector DNA and protoplasts are mixed with 40% polyethylene glycol solution (polyethlene glycol) solution and reacted at room temperature for 20 minutes, and cultured at a temperature of 20 to 30 ° C. for 15 to 17 hours in a CKMM liquid medium and collected. Can be.

상기 형질전환체의 균주는 기계충버섯(Irpex lacteus) 또는 아교버섯(Phlebia tremellosa)일 수 있다.The strain of the transformant may be a machine mushroom ( Irpex lacteus ) or gluco mushroom ( Plebia tremellosa ).

상기 환경호르몬은 비스페놀에이(Bisphenol A) 또는 부틸펜질프탈레이트(butylbenzyl phthalate)일 수 있다.The environmental hormone may be bisphenol A (Bisphenol A) or butylbenzyl phthalate.

이하 본 발명인 2종의 리그닌 분해효소 발현 형질전환체, 그의 제조방법 및 이를 이용한 환경호르몬을 분해하는 방법에 대하여 상세하게 설명한다. 그러나 이는 본 발명을 예시하기 위한 것으로 본 발명의 권리범위를 이에 한정하고자 하는 것은 아니다.Hereinafter, two kinds of lignin degrading enzyme-expressing transformants of the present invention, a preparation method thereof, and a method of degrading environmental hormones using the same will be described in detail. However, this is intended to illustrate the present invention and is not intended to limit the scope of the present invention.

<실시예 1> 사용 균주(菌株) 의 배양(培養)Example 1 Cultivation of Strains Used

기계충버섯(Irpex lacteus), 아교버섯(Phlebia tremellosa)을 공시 균주로 사용하였다. 공시 균주는 리그닌 분해효소 활성을 증가시키기 위해 본래의 박층고정상(SSC) 배지를 약간 변화시켜 사용하였다.Machine mushrooms ( Irpex lacteus ) and glue mushrooms ( Plebia tremellosa ) were used as the disclosed strain. The disclosed strain was used with a slight change in the original thin layered phase (SSC) medium to increase lignin degrading enzyme activity.

본 실험에서 사용된 액체배지 1 L에는 glucose 10 g, (NH4)2C4H4O6 0.5 g, Thiamine-HCl 0.5 mg, KH2PO4 0.5 g, MgSO47H2O 0.5 g, Ca(H2PO4)2 0.5 g, 그리고 소량의 무기질 혼합물이 첨가되었으며, pH는 4.5로 조정하였다. In 1 L of the liquid medium used in this experiment, glucose 10 g, (NH 4 ) 2 C 4 H 4 O 6 0.5 g, Thiamine-HCl 0.5 mg, KH 2 PO 4 0.5 g, MgSO 4 7H 2 O 0.5 g, Ca 0.5 g (H 2 PO 4 ) 2 , and a small amount of mineral mixture were added and the pH was adjusted to 4.5.

상기 무기질 혼합물은 MgSO4 3 g, NaCl 1 g, MnSO4 0.5 g, FeSO47H2O 0.1 g, CoCl2 0.1 g, ZnSO47H2O 0.1 g, CuSO4 0.1 g, AlK(SO4)212H2O 10 mg, nitriloacetic acid 1.5 g을 1 L의 증류수에 첨가하여 0.2 ㎛ 여과지를 이용하여 여과 후 배지에 첨가한 것이다.The inorganic mixture is MgSO 4 3 g, NaCl 1 g, MnSO 4 0.5 g, FeSO 4 7H 2 O 0.1 g, CoCl 2 0.1 g, ZnSO 4 7H 2 O 0.1 g, CuSO 4 0.1 g, AlK (SO 4 ) 2 10 mg of 12H 2 O and 1.5 g of nitriloacetic acid were added to 1 L of distilled water, followed by filtration using 0.2 μm filter paper and then added to the medium.

접종은 PDA 배지에서 7 일간 선 배양된 균을 대상으로 8 ㎜ 직경의 plug을 제조한 후 배지 20 ㎖ 당 1 개의 plug을 접종하였으며 25℃에서 진탕배양을 수행하였다.Inoculation was performed for 7 days pre-cultivated bacteria in PDA medium 8 mm diameter plug was prepared inoculated one plug per 20 ml medium and cultured at 25 ℃ shaking.

<실시예 2> 제조된 균주의 원형질(原形質) 분리<Example 2> Plasma isolation of the prepared strain

10일간 PDA 배지에서 배양한 공시균주의 균사를 수술용 칼로 잘라 PDB 배지 100 ㎖에 넣고 5 일간 1차 배양하였다. 배양 5 일째 자란 균사체를 blending하여, PDB 배지 100 ㎖에 20% (V/V) 농도로 2차 배양하였다. Mycelial strains of the strains cultured in PDA medium for 10 days were cut with a surgical knife and placed in 100 ml of PDB medium, followed by primary culture for 5 days. Mycelia grown on the 5th day of culture were blended and secondarily incubated in 100 ml of PDB medium at 20% (V / V) concentration.

5일 후 같은 방법으로 blending한 균사체를 20% (V/V) 농도로 하여 CKMM 최소배지 100 ㎖에 넣고 1일 배양하였다. After 5 days, the blended mycelium was added to 100 ml of CKMM medium at 20% (V / V) concentration and cultured for 1 day.

상기 CKMM 최소배지는 1% 말토오즈(maltose), 0.2% KNO3, 미량원소(trace element) 40 ㎖, 염액(salt solution) 25 ㎖, 0.1 ㎎/㎖의 티아민 용액(thiamine solution) 1 ㎖로 구성된다.The CKMM minimum medium consists of 1% maltose, 0.2% KNO 3 , 40 ml of trace elements, 25 ml of salt solution, and 1 ml of 0.1 mg / ml thiamine solution. do.

상기 미량원소는 CaCl2, MgCl26H2O, FeCl36H2O, citric acid, MnSO4, ZnSO4로 구성된다.The trace element is composed of CaCl 2 , MgCl 2 6H 2 O, FeCl 3 6H 2 O, citric acid, MnSO 4 , ZnSO 4 .

상기 염액은 ammonium tartrate, KH2PO4, Na2PO4, Na2HPO4, Na2SO4로 구성된다.The salt solution is composed of ammonium tartrate, KH 2 PO 4 , Na 2 PO 4 , Na 2 HPO 4 , Na 2 SO 4 .

상기와 같이 배양하여 얻은 균사체를 원심분리 (Hettich Rotina 38R, 3,000 rpm, 10 min, 25 ℃)하여 회수하였다.The mycelia obtained by culturing as described above were recovered by centrifugation (Hettich Rotina 38R, 3,000 rpm, 10 min, 25 ° C).

상기와 같이 회수한 균사체는 삼투압 안정제 (0.55 M sucrose)를 이용하여 2번 세척(washing)하고, 원심분리 (Hettich Universal 320R, 3,000 rpm, 10 min, 25 ℃)했다. The mycelia recovered as above were washed twice with an osmotic stabilizer (0.55 M sucrose) and centrifuged (Hettich Universal 320R, 3,000 rpm, 10 min, 25 ° C.).

원형질체 생성을 위해 원형질체 생성효소로서 1% Novozyme 234를 회수한 균사체양에 대해 2배양의 삼투압안정제에 녹여서 균체에 첨가하고 이것을 30 ℃, 70 rpm로 VISION사의 쉐이킹 인큐베이터(shacking incubator)에서 1시간 동안 흔들어 주면서 원형질체가 형성되는 것을 확인하였다. For the production of protoplasts, 1% Novozyme 234 recovered as a protoplast generating enzyme was dissolved in an osmotic stabilizer in two cultures and added to the cells. It was confirmed that protoplasts were formed while giving.

상기와 같이 형성된 원형질체는 Aldrich chemical사의 필트레이션Protoplasts formed as described above are the filtration of Aldrich Chemical

어셈블리(filtration Assembly)를 이용하여 회수하였다.Recovery was performed using an filtration assembly.

상기와 같이 회수된 원형질체는 STC 버퍼(buffer)로 2번 세척단계를 거쳐 (Hettich Universal 320R, 9,000 rpm, 20 min, 4 ℃) 반응에 관련된 효소를 제거하였다. 상기 STC 버퍼는 0.55 M Sorbitol, 10 mM Tris HCl, 10 mM CaCl2로 구성된다.The protoplasts recovered as above were washed twice with STC buffer (Hettich Universal 320R, 9,000 rpm, 20 min, 4 ° C.) to remove enzymes involved in the reaction. The STC buffer consists of 0.55 M Sorbitol, 10 mM Tris HCl, 10 mM CaCl 2 .

최종적으로 원형질체가 약 1.0×107/㎖가 되도록 STC 버퍼에 현탁하였다.Finally, the protoplasts were suspended in STC buffer so as to be about 1.0 × 10 7 / ml.

<실시예 3> Mn-peroxidase 및 락카아제 동시 발현 형질전환용 벡터의 제조Example 3 Preparation of Mn-peroxidase and Lacase Coexpression Transformation Vector

1단계로 락카아제와 MnP 유전자를 동시발현시키는 발현벡터는 pBARGPE1 벡터를 기초로 사용하여 구축하였다. pBARGPE1이 가진 gpd promoter를 이용하고자 상기 프로모터 다음에 존재하는 제한효소 SmaI, EcoRI으로 vector를 digestion 한 후 서열목록 7과 같은 겨울 우산 버섯 MnP cDNA 유전자(dir 1.1 kb)에 각각 SmaI, EcoRI linker(서열목록 1 및 서열목록 2 참조)를 연결하여 증폭하고, 상기 절단된 벡터와 ligation하여 pBARMnP를 제작하였다(도 1 참조).In the first step, an expression vector co-expressing laccase and MnP gene was constructed based on the pBARGPE1 vector. In order to use the gpd promoter of pBARGPE1, the vector was digested with the restriction enzymes Sma I and Eco RI after the promoter, and the Sma I and Eco RI, respectively, were added to the winter umbrella mushroom MnP cDNA gene (dir 1.1 kb) as shown in SEQ ID NO: 7. A linker (see SEQ ID NO: 1 and SEQ ID NO: 2) was linked and amplified, and ligation with the cleaved vector produced pBARMnP (see Figure 1).

2단계로 제작된 벡터에 다시 ‘서열목록 5와 같은, 아교버섯으로부터 수득한 프로모터(promoter)’ 및 ‘laccase genomic DNA(약 3.3 kb)’를 삽입하고자 다음의 실험을 진행하였다. 각각 서열목록 3과 같은 Forward primer와 서열목록 4와 같은 Reverse primer를 XhoI linker로 연결한 laccase genomic DNA의 primer를 사용하여 전체 유전자를 증폭하고 Xho1으로 절단한 후 동일 효소로 자른 pBARMnP 벡터에 삽입하여 pMnP-gLac 발현벡터를 구축하였다(도 3 참조).The following experiment was carried out to insert the promoter obtained from the glial mushroom, such as 'SEQ ID NO: 5' and the 'laccase genomic DNA (about 3.3 kb)', into the vector prepared in the second step. Using a primer of laccase genomic DNA linked with a forward primer like SEQ ID NO: 3 and a reverse primer like SEQ ID NO: 4 using Xho I linker, amplify the whole gene, cut it with Xho 1, and insert it into the pBARMnP vector cut with the same enzyme. PMnP-gLac expression vector was constructed (see FIG. 3).

<실시예 4> Mn-peroxidase 및 락카아제 동시 발현 형질전환용 벡터의 확인 실험<Example 4> Confirmation experiment of vector for transformation expression of Mn-peroxidase and laccase

1단계로 pBARGPE 기본벡터에 겨울우산버섯의 MnP 유전자를 삽입하여 gpd 프로모터에 의해 MnP가 과발현하는 6.6 kb 크기의 형질전환벡터 pBARMnP를 제작하고 PCR로 확인하였다(도 1 및 도 2 참조).In step 1, the MnP gene of the winter umbrella was inserted into the pBARGPE base vector to prepare a transformed vector pBARMnP having a size of 6.6 kb in which MnP was overexpressed by the gpd promoter and confirmed by PCR (see FIGS. 1 and 2).

2단계로 형질전환벡터 pBARMnP에 다시 아교버섯의 laccase gDNA를 삽입하여 MnP 유전자 및 laccase 유전자가 동시에 발현하는 9.9 kb 크기의 pMnP-gLac 벡터 제작하고 PCR로 확인하였다(도 3 및 도 4 참조).In step 2, the laccase gDNA of the glial mushroom was inserted again into the transformation vector pBARMnP to prepare a 9.9 kb pMnP-gLac vector expressing the MnP gene and the laccase gene at the same time, and confirmed by PCR (see FIGS. 3 and 4).

<실시예 5> 형질전환(形質轉換)Example 5 Transformation

제한효소 매개 삽입법(Restriction enzyme mediated integration)에 의한 형질전환을 위해 5 ㎍의 벡터 DNA를 제한효소로 절단하여 STC buffer에 현탁되어 있는 300 ㎕의 원형질체 (1 x 107개 / ㎖)에 넣었다.For transformation by restriction enzyme mediated integration, 5 μg of vector DNA was digested with restriction enzymes and placed in 300 μl of protoplasts (1 × 10 7 pieces / ml) suspended in STC buffer.

이후 40% PEG(polyethlene glycol) 용액을 섞어서 상온에서 20분 반응하고 삼투압 안정제로써 0.55 M sorbitol이 첨가된 CKMM 액체배지에서 25℃에서 16 시간정도 배양하였다. Then, 40% PEG (polyethlene glycol) solution was mixed and reacted at room temperature for 20 minutes, and incubated at 25 ° C. for 16 hours in a CKMM liquid medium to which 0.55 M sorbitol was added as an osmotic stabilizer.

상기 PEG용액은 40% 폴리에틸렌글리콜(polyethlene glycol)4000, 25 mM CaCl2, 25 mM Tris-HCl으로 이루어진 pH 7.5의 용액이다.The PEG solution is a solution of pH 7.5 consisting of 40% polyethylene glycol (polyethlene glycol) 4000, 25 mM CaCl 2 , 25 mM Tris-HCl.

상기 액체배지에서 배양된 혼합물 100 ㎕를 0.55 M sorbitol과 0.8% agar를 포함한 10 ㎖ CKMM 선별배지에 섞고 이를 bottom 배지인 CKMM 선별배지에 부어 25℃에서 배양하였다.100 μl of the mixture incubated in the liquid medium was mixed in a 10 ml CKMM selection medium containing 0.55 M sorbitol and 0.8% agar and poured into the bottom medium CKMM selection medium and incubated at 25 ° C.

상기 bottom 배지는 agar 1.6%이고, 상기 CKMM 선별배지는 0.55 M 솔비톨(sorbitol)을 포함한다.The bottom medium is 1.6% agar and the CKMM selection medium contains 0.55 M sorbitol.

상기 CKMM 선별배지에는 사용하는 균과 벡터에 맞는 항생제로 50 ㎍/㎖ 포스피노트리신(phosphinothricin)과 암피실린(ampicillin)을 각각 첨가하였다. 배양 다음날부터 관찰하여 균사가 형성되는 것이 보이기 시작하면 균사 부분만 칼로 잘라서 항생제의 농도를 두 배로 증가시킨 2차 선별배지로 옮겨 배양하였다.To the CKMM selection medium, 50 μg / ml phosphinothricin and ampicillin were added as antibiotics suitable for the bacteria and vectors used. When the hyphae began to be observed from the day after cultivation, only the mycelium part was cut with a knife, and the culture was transferred to a secondary screening medium which doubled the concentration of antibiotics.

<실시예 6> 형질전환체의 벡터삽입 여부의 확인 실험Example 6 Confirmation Experiment of Vector Insertion of Transformant

항생제 배지에서 선발된 형질전환체의 균사로부터 지노믹 디엔에이(genomic DNA)를 분리하여 형질전환에 사용한 벡터의 염기서열을 바탕으로 제작된 primer 쌍을 통한 PCR을 수행하여 유전자의 삽입 유무를 확인하였다.Genomic DNA was isolated from the mycelia of the transformant selected from antibiotic medium, and PCR was performed through a primer pair prepared based on the nucleotide sequence of the vector used for transformation.

PDB(Potato Dextrose Broth)에서 키운 형질전환체 또는 대조구로부터 균체를 여과지로 모은 후 1.5 ㎖ 1.5 ㎖ 튜브(tube)에 100 ㎎ 정도 담고, DNA extraction buffer (0.2 M Tris-Hcl (pH 8) 80 ㎖, 0.25 M NaCl, 0.025 M EDTA (pH 8), 0.5% SDS)를 100 ㎕ 넣고 일회용 플라스틱 막자로 갈아준 다음 13,000 rpm으로 4℃에서 15분간 원심분리 시켰다. 분리된 상등액을 새 tube에 옮긴 후, 페놀(phenol)과 클로로폼(chloroform)을 상등액의 1/2씩 넣어 섞은 후 13,000 rpm으로 상온(약 25℃)에서 15분 원심분리 시켜 깨끗한 상등액만 새 튜브로 옮기고 이소프로판올(isopropanol)을 상층액의 70%를 넣고 부드럽게 섞은 다음 13,000 rpm으로 4℃에서 20분간 원심분리하여 상등액은 버린 후 70% 에탄올(ethanol)을 500 ㎕ 넣고 5분간 다시 원심분리하여 genomic DNA를 얻었다. Cells were collected from transformants or controls grown in PDB (Potato Dextrose Broth) with filter paper and placed in 100 ml in 1.5 ml 1.5 ml tubes, 80 ml of DNA extraction buffer (0.2 M Tris-Hcl (pH 8)), 100 μl of 0.25 M NaCl, 0.025 M EDTA (pH 8), 0.5% SDS) was added to a disposable plastic pestle, and centrifuged at 13,000 rpm for 15 minutes at 4 ° C. Transfer the separated supernatant to a new tube, mix phenol and chloroform 1/2 of the supernatant, mix them, and centrifuge at 13,000 rpm for 15 minutes at room temperature (about 25 ℃). Transfer the isopropanol to 70% of the supernatant, mix gently, and centrifuge at 13,000 rpm for 20 minutes at 4 ° C. Discard the supernatant, remove 500% of 70% ethanol and centrifuge again for 5 minutes. Got.

분리된 지노믹 디엔에이는 1 ㎕로 벡터에 특이적인 forward primer(Bar G; 5' - CTA CCA TGA GCC CAG AAC G - 3')와 reverse primer(Bar G; 5' - GGG TCA TCA GAT CTC GGT G - 3')를 이용하여 PCR (1 step, 1cycle; 95℃, 5 min, 2 step, 25 cycle; 95℃, 1 min; 60℃, 2 min; 72℃, 2 min, 3 step, 1cycle; 72°C, 5 min)을 수행하여 전기영동한 후 PCR 산물의 크기를 확인하였다.The isolated genomic DNA was 1 μl and the vector-specific forward primer (Bar G; 5 '-CTA CCA TGA GCC CAG AAC G-3') and reverse primer (Bar G; 5 '-GGG TCA TCA GAT CTC GGT G 3 ') using PCR (1 step, 1 cycle; 95 ° C., 5 min, 2 step, 25 cycles; 95 ° C., 1 min; 60 ° C., 2 min; 72 ° C., 2 min, 3 step, 1cycle; 72 ° C, 5 min) was carried out by electrophoresis to determine the size of the PCR product.

기계충버섯 및 아교버섯을 각각 형질전환용 벡터인 pMnP-gLac로 형질전환하여 PCR 했을때 약 560 bp 크기의 PCR 산물을 확인할 수 있었다(도 5 및 도 6 참조). When the machine insect mushroom and the glue mushroom were transformed with pMnP-gLac, which is a transformation vector, respectively, PCR products of about 560 bp size were identified (see FIGS. 5 and 6).

그러나 음성 대조구로 사용된 기계충버섯 및 아교버섯의 야생형 균주에서는 같은 위치에 PCR 산물이 나타나지 않았다. 따라서 형질전환체에 pMnP-gLac 벡터의 플라스미드가 균사체의 게놈 유전자 내로 성공적으로 삽입되었음을 알 수 있었다.However, wild-type strains of machine mushrooms and glue mushrooms used as negative controls did not show PCR products. Therefore, the plasmid of the pMnP-gLac vector was successfully inserted into the genomic gene of the mycelia.

<실시예 7> 환경호르몬(EDC; Endocrine Disrupters) 분해능 분석Example 7 Analysis of Endocrine Disrupters (EDC) Resolution

PDA배지에서 선배양 된 형질전환체와 야생균주의 균사 2 plug을 접종하여 10일 동안 배양 후, PDB 배지에 BPA(Bisphenol A) 100 ppm 또는 BBP(Benzylbutylphthalate) 300 ppm, 을 첨가하여 무처리군과 함께 7일간 배양하였다.After inoculating 2 plugs of hyphae with transformants and wild strains pre-cultured in PDA medium, and incubated for 10 days, 100 ppm of BPA (Bisphenol A) or 300 ppm of BBP (Benzylbutylphthalate) was added to the PDB medium. Incubated together for 7 days.

배양액의 상등액을 얻기 위하여 9,000 rpm에서 40 분 동안 cell down 시켰다. o-tolidin이 효소에 의해 산화되어 발색되는 정도를 흡광도로 측정하기위하여, 이 상등액의 7.5 ㎕을 취하여 o-tolidine 300 ㎕과 섞은 후 실온에서 30분 반응을 시켜 595 nm에서 발색되는 정도를 흡광도(microplate spectrophtometer)로 측정하였다. Cell down was performed for 40 minutes at 9,000 rpm to obtain supernatant of the culture. To measure the degree of o- tolidin oxidization and color development by enzymes, take 7.5 μl of this supernatant and mix it with 300 μl of o- tolidine, and react for 30 minutes at room temperature to absorb the color at 595 nm. microplate spectrophtometer).

그리고 환경호르몬 잔존량을 확인하기 위하여 야생균주와 형질전환체의 남은 상등액 35 ㎖과 헥세인(Hexanes) 20 ㎖를 넣고 1차 추출하고 헥세인 : 에틸 아세테이트(Ethly Acetate) 20 ㎖ 넣어 2차 추출을 하였다. 2차 추출액에 Na2SO4를 넣고 불순물을 제거한 뒤 여과지(filter paper)로 여과 하였으며, 이 추출액 4 ㎖를 질소로 농축 시키고 추출액이 다 날아간 용기에 Hexanes 400 ㎕을 첨가하여 녹여준 후 HPLC(Waters 1525 binary pump) 분석을 통하여 환경호르몬 잔존량을 조사하였다. 또한 이스트 투 하이브리드 시스템(Yeast two hybrid system)으로 환경호르몬 분해산물의 에스트로겐 활성을 확인하기 위하여 에스트로겐성 관련 유전자 발현을 확인할 수 있는 재조합 효모를 SD배지(Minimal SD base, Lea, Trp DO supplement(Clontech)) 2 ㎖에 배양 (30℃ 24시간)을 한 뒤 50㎕를 취하여 선배양된 형질전환체와 야생균주 배양액에 EDC를 넣었다. EDC 투입 직후의 배양액, EDC 투입 1일, 3일, 5일, 7일 간격으로 자란 배양균의 배양액과 각 시기별 무처리군 배양액 20 ㎕을 재조합 효모가 배양된 200 ㎕의 SD배지에 접종하여 30℃에서 4시간을 다시 배양하였다. In order to confirm the remaining amount of environmental hormones, 35 ml of the remaining supernatant of the wild strain and the transformant and 20 ml of hexane were added, followed by primary extraction and 20 ml of hexane: ethyl acetate (Ethly Acetate). It was. Na 2 SO 4 was added to the secondary extract, and impurities were removed and filtered through a filter paper. 4 ml of the extract was concentrated with nitrogen, and 400 μl of Hexanes was added to the container where the extract had flown to dissolve, followed by HPLC (Waters). 1525 binary pump) was used to investigate environmental hormone levels. In addition, the yeast two hybrid system (SD Mini, base, Lea, Trp DO supplement (Clontech)) is a recombinant yeast capable of confirming estrogen-related gene expression in order to confirm estrogen activity of environmental hormone degradation products. Incubated in 2 ml (30 ° C. for 24 hours), 50 μl was taken and EDC was added to the pre-cultured transformant and wild strain culture. Immediately after the EDC addition, 20 μl of the culture medium grown at 1, 3, 5, and 7 days after the EDC addition and 20 μl of the untreated group culture at each time period were inoculated into 200 μl of SD medium cultured with recombinant yeast. Incubated 4 hours again at 30 ℃.

상기와 같은 배양 후 10분 동안 원심분리(15,000 rpm, 25℃)하여 상등액을 버리고 pellet을 취하여 200 ㎕의 Z Buffer(0.1M sodium phosphate pH 7.0, 10 mM KCl, 1 mM MgSO4, 0.001% SDS)에 녹였다. 여기에 4㎕의 클로로폼(chloroform)을 첨가 하고 잘 섞어준 후 30℃에서 15분 동안 반응을 시켜주었다. 40㎕의 ONPG(o-nitrophenyl-D-galactopyranoside)를 첨가하여 반응액이 엷은 노란색으로 변하는 시점(2?3일)에서 반응을 멈추고 470 nm에서 흡광도를 측정하였다.Discard the supernatant by centrifugation (15,000 rpm, 25 ℃) for 10 minutes after the incubation as described above and take pellet to 200 μl Z Buffer (0.1M sodium phosphate pH 7.0, 10 mM KCl, 1 mM MgSO 4 , 0.001% SDS) Dissolved in. 4 μl of chloroform was added and mixed well, followed by reaction at 30 ° C. for 15 minutes. 40 µl of ONPG (o-nitrophenyl-D-galactopyranoside) was added to stop the reaction at the point where the reaction solution turned pale yellow (2-3 days), and the absorbance was measured at 470 nm.

<실시예 8> 기계충버섯 및 아교버섯 형질전환체의 BBP 분해 실험Example 8 BBP Degradation of Machine Mushroom and Glue Mushroom Transformants

형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BBP을 넣고 배양한 후, HPLC로 분해된 환경호르몬 잔존량을 조사하였다. In order to investigate the environmental hormone degrading ability by the transformant, BBP was added to the culture medium and cultured, and the residual amount of environmental hormones degraded by HPLC was examined.

그 결과 야생균주보다 기계충버섯 및 아교버섯에 의해 환경호르몬 잔존량이 감소하였다. As a result, the residual amount of environmental hormones was reduced by the mushrooms and glue mushrooms than wild strains.

특히, 기계충버섯은 BBP를 넣고 배양 24시간 경과 후(도 7 참조), 아교버섯 은 48시간 경과 후 야생균주에 비해 현저히 BBP잔존량이 감소하였다(도 8 참조).In particular, after 24 hours of culture (see Fig. 7), the mechanical mushroom mushrooms BBP after the 48 hours, the residual BBP significantly decreased compared to the wild strain (see Fig. 8).

<실험예 10> 기계충버섯 및 아교버섯 형질전환체의 BPA 분해 실험Experimental Example 10 BPA Degradation Experiment of Mechanical Mushroom and Glue Mushroom Transformants

형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BPA를 넣고 배양한 후 HPLC로 분해된 환경호르몬 잔존량을 조사하였다. In order to investigate the environmental hormone degrading ability by the transformant, BPA was added to the culture medium, and the residual amount of environmental hormone decomposed by HPLC was examined.

그 결과 야생균주보다 기계충버섯 및 아교버섯에 의해 환경호르몬 잔존량이 감소하였다. As a result, the residual amount of environmental hormones was reduced by the mushrooms and glue mushrooms than wild strains.

다만, BPA를 넣고 배양한 경우 BBP를 넣고 배양한 경우와 비교하여 상대적으로 감소량이 적은 것으로 나타났다(도 9 및 도 10 참조).However, in the case of cultured with BPA, it was found that the amount of reduction was relatively small compared to the culture with BBP (see FIGS. 9 and 10).

<실시예 11> 기계충버섯의 형질전환체를 통한 분해산물의 에스트로겐성 실험<Example 11> Estrogenity test of the degradation product through the transformant of the machine mushroom

환경호르몬의 에스트로겐성을 보여줄 수 있는 이스트 투 하이브리드 시스템(Yeast two hybrid system)으로 환경호르몬 분해산물의 에스트로겐 활성을 확인하였다. The yeast two hybrid system, which can show the estrogen properties of environmental hormones, confirmed the estrogen activity of the degradation products of environmental hormones.

즉, 기계충버섯의 형질전환체를 환경호르몬인 BBP와 함께 배양한 후 분해산물의 에스트로겐성을 측정한 결과 72시간 경과 시 야생균주에 비해 에스트로겐성이 현저히 줄어들어 약 90%의 감소율을 보였다(도 11 참조).In other words, after transforming the transformant of mechanical insect mushroom with BBP, an environmental hormone, the estrogenity of the degradation product was measured, and the estrogenity was significantly decreased compared to wild strains after 72 hours. 11).

또 다른 환경호르몬인 BPA를 형질전환체와 함께 배양했을 때는 BBP보다 에스트로겐성이 적게 줄어드는 경향을 보임으로써(도 13 참조) 형질전환체가 BPA보다 BBP를 더 잘 분해함을 알 수 있었다.When BPA, another environmental hormone, was cultured with a transformant, it showed a tendency to decrease estrogenity less than that of BBP (see FIG. 13), indicating that the transformant decomposed BBP better than BPA.

즉 pMnP-gLac로 형질전환하여 MnP 유전자 및 laccase 유전자가 동시에 발현하는 기계충버섯은 야생균주에 비해 환경호르몬을 잘 분해하며 BPA보다는 BBP를 더 잘 분해하는 것으로 나타났다.In other words, mechanical mushrooms transformed with pMnP-gLac and expressing both MnP and laccase genes were found to degrade environmental hormones better than wild strains, and to degrade BBP better than BPA.

<실시예 12> 아교버섯의 형질전환체를 통한 분해산물의 에스트로겐성 실험<Example 12> Estrogenity test of the degradation product through the transformant of the gluco mushroom

상기 실험례 ~와 같이 이스트 투 하이브리드 시스템을 통해 환경호르몬 분해산물의 에스트로겐 활성을 확인하였다. Experimental Example As shown in the yeast-to-hybrid system was confirmed the estrogen activity of the environmental hormone degradation products.

즉, 아교버섯의 형질전환체를 환경호르몬인 BBP와 함께 배양한 후 분해산물의 에스트로겐성을 측정한 결과 72시간 경과 시 야생균주에 비해 에스트로겐성이 현저히 줄어들어 약 80%의 감소율을 보였다(도 12 참조).That is, after culturing the transformant of the gluco mushroom with BBP, an environmental hormone, the estrogenity of the degradation product was measured, and the estrogenity was significantly decreased after 72 hours compared to the wild strain, resulting in a decrease of about 80% (FIG. 12). Reference).

또 다른 환경호르몬인 BPA를 형질전환체와 함께 배양했을 때는 BBP보다 에스트로겐성이 적게 줄어드는 경향을 보임으로써(도 14 참조) 형질전환체가 BPA보다 BBP를 더 잘 분해함을 알 수 있었다.When BPA, another environmental hormone, was incubated with a transformant, it showed a tendency to decrease estrogenity less than BBP (see FIG. 14), indicating that the transformant degraded BBP better than BPA.

즉 아교버섯의 경우에도 pMnP-gLac로 형질전환하여 MnP 유전자 및 laccase 유전자가 동시에 발현하도록 한 결과, 야생균주에 비해 환경호르몬을 잘 분해하며 BPA보다는 BBP를 더 잘 분해하는 결론을 얻을 수 있었다.In other words, even in the case of glucose mushrooms, pMnP-gLac was transformed to express MnP gene and laccase gene at the same time. As a result, it was concluded that the degradation of environmental hormones was better than that of wild strains and BBP was better than BPA.

종래 백색부후균의 형질전환체는 락카아제(laccase) 또는 망간 퍼옥시다아제(Mn peroxidase) 하나의 유전자만을 삽입한 형질전환체로서 리그닌 및 리그닌과 구조가 유사한 환경호르몬의 생물학적 분해능을 실질적으로 미흡한 단점이 있었다.Conventional white fungal transformants are laccase or manganese peroxidase transformants that have only one gene inserted, which has a disadvantage in that they substantially lack the biological resolution of environmental hormones similar in structure to lignin and lignin. there was.

상기 문제점 해결하기 위해서 본원 발명은 환경호르몬을 분해하는 우수 균주 중 기계충버섯과 아교버섯을 선택하여 리그닌 분해효소 중 락카아제 및 망간 퍼옥시다아제 유전자가 동시에 발현하는 재조합 벡터를 도입한 형질전환체를 제공한다.In order to solve the above problems, the present invention provides a transformant by introducing a recombinant vector which simultaneously expresses the lactose and manganese peroxidase genes in lignin degrading enzymes by selecting a machine mushroom and a glucocorticoid among excellent strains that degrade environmental hormones. do.

상기 형질전환체를 이용함으로써 리그닌 또는 리그닌과 구조가 유사한 비스페놀 A와 벤질부틸프탈레이트 등과 같은 환경호르몬 분해능을 극대화시킬 수 있다.By using the transformant, it is possible to maximize environmental hormone resolution, such as bisphenol A and benzylbutyl phthalate, which are similar in structure to lignin or lignin.

따라서 최근 보편화된 일회용 용기 등의 사용으로 인한 불임, 기형아 출산 또는 암 등의 환경호르몬 관련 질병을 해소할 수 있다는 점에서 본원 발명은 산업상이용가능성이 매우 우수하며, 궁극적으로는 국가의 친환경적 생명공학 산업발전에 이바지 할 것으로 기대된다.Therefore, the present invention has excellent industrial applicability in that it can relieve environmental hormone-related diseases such as infertility, birth defects or cancer caused by the use of the recently-used disposable container, and ultimately, eco-friendly biotechnology of the country. It is expected to contribute to industrial development.

도 1은 pBARGPE 기본벡터에 겨울우산버섯의 MnP 유전자를 삽입하여 gpd 프로모터에 의해 MnP가 과발현하는 6.6 kb 크기의 형질전환벡터 pBARMnP를 제작하는 과정을 간단하게 나타낸 것이다.Figure 1 simply shows the process of preparing a transformed vector pBARMnP having a size of 6.6 kb MnP overexpressed by the gpd promoter by inserting the MnP gene of the winter umbrella in the pBARGPE base vector.

도 2는 pBARGPE 기본벡터에 겨울우산버섯의 MnP 유전자를 삽입하여 gpd 프로모터에 의해 MnP가 과발현하는 6.6 kb 크기의 형질전환벡터 pBARMnP를 제작한 것을 PCR로 확인한 사진이다.Figure 2 is a PCR confirmed that a 6.6 kb transformed vector pBARMnP of MnP overexpressed by the gpd promoter by inserting the MnP gene of the winter umbrella in pBARGPE base vector.

도 3은 형질전환벡터 pBARMnP에 다시 아교버섯의 laccase gDNA를 삽입하여 MnP 유전자 및 laccase 유전자가 동시에 발현하는 9.9 kb 크기의 pMnP-gLac 벡터 제작하는 과정을 간단하게 나타낸 것이다.Figure 3 simply shows the process of preparing a 9.9 kb sized pMnP-gLac vector expressing the MnP gene and laccase gene at the same time by inserting the laccase gDNA of the glial mushroom into the transformation vector pBARMnP.

도 4는 형질전환벡터 pBARMnP에 다시 아교버섯의 laccase gDNA를 삽입하여 MnP 유전자 및 laccase 유전자가 동시에 발현하는 9.9 kb 크기의 pMnP-gLac 벡터 제작한 것을 PCR로 확인한 사진이다.FIG. 4 is a PCR photograph showing that a 9.9 kb-sized pMnP-gLac vector, in which a MlP gene and a laccase gene are simultaneously expressed by inserting a laccase gDNA of a glial mushroom into the transformation vector pBARMnP, is shown.

도 5는 기계충버섯을 각각 형질전환용 벡터인 pMnP-gLac로 형질전환하여 PCR 했을때의 약 560 bp 크기의 PCR 산물을 확인한 사진이다.5 is a photograph showing the PCR product of about 560 bp when the machine mushroom mushrooms were transformed with pMnP-gLac, which is a transformation vector, respectively.

도 6는 아교버섯을 각각 형질전환용 벡터인 pMnP-gLac로 형질전환하여 PCR 했을때의 약 560 bp 크기의 PCR 산물을 확인한 사진이다.Figure 6 is a photograph showing a PCR product of about 560 bp when transformed by PCR with pMnP-gLac, a transformation vector, respectively.

도 7은 기계충버섯의 형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BBP을 넣고 배양한 후, HPLC로 분해된 환경호르몬 잔존량을 조사한 그래프이다.FIG. 7 is a graph illustrating the amount of environmental hormone remaining after degradation by HPLC after incubating BBP in a culture medium to investigate the ability to degrade environmental hormones by transformants of the machine mushroom.

도 8은 아교버섯의 형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BBP을 넣고 배양한 후, HPLC로 분해된 환경호르몬 잔존량을 조사한 그래프이다.FIG. 8 is a graph illustrating the amount of environmental hormone remaining after degradation by HPLC after incubating BBP in a culture medium to investigate the ability of environmental hormone degradation by a transformant of a gluco mushroom.

도 9은 기계충버섯의 형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BPA를 넣고 배양한 후, HPLC로 분해된 환경호르몬 잔존량을 조사한 그래프이다.FIG. 9 is a graph illustrating the amount of environmental hormone remaining after degradation by HPLC after incubating with BPA in a culture medium to investigate the ability of environmental hormone degradation by the transformant of the mechanical mushroom. FIG.

도 10은 아교버섯의 형질전환체에 의한 환경호르몬 분해력을 조사하기위해 배양액에 BPA를 넣고 배양한 후, HPLC로 분해된 환경호르몬 잔존량을 조사한 그래프이다.FIG. 10 is a graph illustrating the amount of environmental hormone remaining after degradation by HPLC after incubating BPA in a culture medium to investigate the ability of environmental hormone degradation by a transformant of a gluco mushroom.

도 11은 기계충버섯 형질전환체에 의한 BBP 분해산물의 에스트로겐성 분석을 위해 이스트 투 하이브리드 시스템(Yeast two hybrid system)으로 환경호르몬 분해산물의 에스트로겐 활성을 확인한 결과이다.11 is a result of confirming the estrogen activity of the environmental hormone degradation products in the yeast two hybrid system (Yeast two hybrid system) for the estrogen analysis of BBP degradation products by the machine mushroom transformant.

도 12는 아교버섯 형질전환체에 의한 BBP 분해산물의 에스트로겐성 분석을 위해 이스트 투 하이브리드 시스템으로 환경호르몬 분해산물의 에스트로겐 활성을 확인한 결과이다.12 is a result of confirming the estrogen activity of the environmental hormone degradation products in the yeast two hybrid system for the estrogen analysis of BBP degradation products by the gluco mushroom transformant.

도 13은 기계충버섯 형질전환체에 의한 BPA 분해산물의 에스트로겐성 분석을 위해 이스트 투 하이브리드 시스템으로 환경호르몬 분해산물의 에스트로겐 활성을 확인한 결과이다.Figure 13 is a result of confirming the estrogen activity of the environmental hormone degradation products in the yeast two hybrid system for estrogen analysis of BPA degradation products by the machine mushroom transformant.

도 14은 아교버섯 형질전환체에 의한 BPA 분해산물의 에스트로겐성 분석을 위해 이스트 투 하이브리드 시스템으로 환경호르몬 분해산물의 에스트로겐 활성을 확인한 결과이다.14 is a result of confirming the estrogen activity of the environmental hormone degradation products in the yeast two hybrid system for the estrogen analysis of BPA degradation products by the gluco mushroom transformant.

도 15는 겨울 우산 버섯 발현 벡터 모식도를 나타낸 것이다.Figure 15 shows a winter umbrella mushroom expression vector schematic.

도 16는 아교 Laccase 발현 벡터 모식도를 나타낸 것이다.16 shows a schematic representation of the glue Laccase expression vector.

<110> Korea(Korea Forest Research Institute) <120> Transformant expressing two kinds of ligninases, method for preparing the transformant and method for degrading endocrine disruptor by using the transformant <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Foward primer Sma I linker of Polyporellus brumalis <400> 1 cccgggatgg ccttcaaaac a 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer EcoR I linker of Polyporellus brumalis <400> 2 gaattcttag gagggaggaa cagg 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Foward primer Xho I linker containing Merulius tremellosus promoter <400> 3 ctcgaggcac gctgttactg cc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer Xho I linker of Merulius tremellosus promoter <400> 4 ctcgagggcg tcagtgaagc c 21 <210> 5 <211> 3153 <212> DNA <213> Artificial Sequence <220> <223> gDNA sequence containing Merulius tremellosus promoter <400> 5 gcacgctgtt actgcctgcg tacgcagccc agtgcaacca tcgcctaaca tctgacagtc 60 tatcagttgc aggacaaccc ttagcgatcg ctttgatgtc tagcgaagta cgaggaaagt 120 cagatgctgt tctatctcct acgcagatgc ggacgagatc cagtggaacg ggtatggctc 180 ccccattccc agcctgttct cagctcacgt cttcgccggt cggagtgggt ctcatctgta 240 agtaagtttt tctatcaaac ttagcccgtc gtctcttatt gaactatcac tagcaaaccc 300 ttctccagcg cgagcaagat ccagacactg gttgctcgtg ggagtcactc ttcagtcgcc 360 ttcagtttgg gtccgtgtgt acgtctgatg gctgccggca ccgaaatttg agacaaggtg 420 ggtcgagctt cacccgatga cacggcctgc gattttgaca ttcaagctta tgttcctgac 480 aggtcatctc acgcccaggt ctgcgctcac gcgatattcg gcaaactcta cagaaaatat 540 acattttctg cgaaggccac gagcggcagg ccatcgggat tgccggacat gaaccagatt 600 ctgtaccgag gtgtacggcc ttgtatatct gacttgctct atttgttgtg gtcagaggta 660 tattaaccag tgagtctcct gcctgtgctc cccagttccc cgctgacaag ggattaccag 720 agggcctctt caccaatgtt ttccttcact cgttctactg cgctcgtcgt cgcaggcctt 780 tccgctggtg ccatcgctgc catcggccct gtgactgact ttcatatcgt caatgcagcc 840 atcgcccccg acggtatctc tcgtcaggct gtgcttgctg agggcacatt ccctggtcct 900 cttattactg gcaacaaggt aggaccaact ccgtgttgtc ttttgttcat ttattcatca 960 tcgtaccgaa aataacaggg cgacaacttt caaatcaatg ttatcgacga gctgaccaat 1020 gctaccatgc tcaagtcgac gacgattgtt agttcacgca atactctatt ccacgctctc 1080 tggtgcttac tcttcacttc agcactggca tggattcttc cagcacggaa cgaactgggc 1140 tgatggtccg gccttcgtca atcaatgtcc tattgctacc gggaattctt tcttgtacaa 1200 cttcaacgca cccgatcaag ccggtttgtt gttttacaag gcttcacgta cattgcgtac 1260 tcacgtggtg atgtcggccc tcaggtacat tctggtatca tagtcatctg tccactcagt 1320 actgtgacgg tttgcgtggt ccttttgtgg tctacgatcc caatgatcct tatgcggatt 1380 tgtacgacgt tgatgacggt aagcattcag ccataccgaa gtaacgtagg ctgatatctg 1440 cgccagattc taccgtcatc acgctggccg actggtatca tacggctgcc cgcctcggcg 1500 gtcctttccc gtaagtttaa acagttcatc gcagacagtc aacgacgtat cgactgacct 1560 ttatctaagc aatgccgaca ctactttgat taatggccta ggcagatgcg gtgaaccggg 1620 cgctcctgtc tctgatctcg ctgttatcaa cgtcgaggct ggcaagcggt atgtccacga 1680 tttcagccat gccattcccc tttgtatgct catttcggtc atttgtatag ttatcgaatg 1740 cgcttggttt cgatctcctg tgactcgtgg ttcaatttca ccatcgatgg acacgatatg 1800 accataattg aagttgatgg cgtcaaccac gagacgttga ccgtagacca aatccagatc 1860 ttcgccgctc agcgttactc tttcatcgta agcattcatt gtttctcgta catggagtca 1920 gactctgatc atgaatatat tatagttgga agcaaaccag cctgtcgata actactggat 1980 tcgcgcaaac ccgggtaccg gcatcactca agggttcgag ggcggcatca actcggccat 2040 tctgcgctat gctggcgctg acgaggttga acctaccact gcgttagttg cgagcacctc 2100 tccgttggcc gagggagatt tgcaccctct agacaaccct gctgccgtat gttcatgtcg 2160 catgtccatt cacaaccgat actgatcgat gctctaccag cctggccaac ccttcccagg 2220 cggcgtcgat tacgctttga atttgaactt gacattcgtc agtctagaaa gtcgtctgtg 2280 tgaaaccaca tctgatgtgc tttgcaggat ggaccgaccc ttaagttcct gatcaatggg 2340 gtgtctttcg tacccccgac agttccggtg ctcctgcaga ttctcagtgg tgcgcagacc 2400 gcacaagatc ttcttcctac tgggagtgtc tactccttgc cctcgaacgc gaccattgaa 2460 ttgagtctcc ctgctggtcc tgcaggcgga cctcatccct tccacttgca cggcgtgagt 2520 ttgcctattc ctaatttgca tatggtatta acttaacctt catatagcac acgttcagtg 2580 tggttcaagg tgcaggacaa acggtgccga actacgtgaa tcctgtccgc cgagatgttg 2640 tcagcatcgg agctgttccc gacaatgtca ccattcgttt cgaggtacga atgcaatcag 2700 tatagatgag agggctcttt ttgaccaaag gtcatcgtta cagaccaaca atcccggccc 2760 atggttcctc cattgccaca tcgactggca cttggatgct ggcttcgccg aggtattcgc 2820 tgaagacatc cctgatatcg cttcggtcaa ccctgtcctt agtgagtcac acacatcatg 2880 cacgggtatc aatcaccatg tactgatgac tgccttagcc gactggtcca atctgtgccc 2940 aatctacgac gctctcgatc cttctgacca gtaaggattg gtattgatat gccgttggtg 3000 gctgtgaacg cggcgatgga caatggacta taagtatttg cagtatacat atatctttca 3060 catcacaagg gggaacagat atacggacta gacttttgga gtactgttgg ctcagtagtg 3120 tagttgatga ggcgtcagtg aagccggatc cac 3153 <210> 6 <211> 1832 <212> DNA <213> Artificial Sequence <220> <223> Laccase cDNA sequence of Merulius tremellosus <400> 6 gggggatagg gggggaggaa gcggagttcc ccgctgacaa ggggttgcca gagggcctct 60 tcaccaatgt tttccttcgc tcgttctact gcgctcgtcg tcgcaggcct tttcgttggt 120 gccatcgccg ccatcggccc tgtgactgac tttcatatcg tcaatgcagc catcgccccc 180 gacggtatct ctcgtcaggc cgtgcttgct gagggcacat tccctggtcc tctcattact 240 ggcaacaagg gcgacaactt ccaaatcaat gttatcgacg agctaaccaa tgctaccatg 300 ctcaagtcga cgacgatcca ctggcatgga ttcttccagc acggaacgaa ctgggctgat 360 ggtccggcct tcgtcaatca atgtcctatt gctaccggga attctttctt gtacaacttc 420 aacgcacccg atcaagccgg tacattctgg tatcatagtc atctgtccac tcagtactgt 480 gacggtttgc gtggtccttt tgtggtctac gatcccaatg atccttatgc ggatttgtac 540 gacgttgatg acgattctac cgtcatcacg ctggccgact ggtatcatac ggctgcccgc 600 ctcggcggtc ctttccccaa tgccgacact actttgatta atggcctggg cagatgcggt 660 gaaccgggcg ctcctgtctc tgatctcgct gttatcaacg tcgaggctgg caagcgttat 720 cgaatgcgct tggtttcgat ctcctgtgac tcgtggttca atttcaccat cgatggacac 780 gatatgacca taattgaagt tgatggcgtc aaccacgaga cgttgaccgt agaccaaatc 840 cagatcttcg ccgctcagcg ttactctttc atcttggaag caaaccagcc tgtcgataac 900 tactggattc gcgcaaaccc gggtaccggc atcactcaag ggttcgaggg cggcatcaac 960 tcggccattc tgcgctatgc tggcgctgac gaggttgaac ctaccactgc gttagttgcg 1020 agcacctctc cgttggccga gggagatttg caccctctag acaaccctgc tgccnctggc 1080 caacccttcc caggcggcgt cgattacgct ttgaatttga acttgacatt cgatggaccc 1140 acccttaagt tcctgatcaa tggggtgtct ttcgtacccc cgacagttcc ggtgctcctg 1200 cagattctca gtggtgcgca gaccgcacaa gatcttcttc ctactgggag tgtctactcc 1260 ttgccctcga acgcgaccat tgaattgagt ctccctgctg gtcctgcagg cggacctcat 1320 cccttccact tgcacggcca cacgttcagt gtggttcaag gtgcaggaca aacggtgccg 1380 aactacgtga atcctgtccg ccgagatgtt gtcagcatcg gagctgttcc cgacaatgtc 1440 accattcgtt tcgagaccaa caatcccggc ccatggttcc tccattgcca catcgactgg 1500 cacttggatg ctggcttcgc cgtggtattc gctgaagaca tccccgatat cgcttcggtc 1560 agccctgtcc ctaccgactg gtccaatctg tgcccaatct acgacgctct cgatccttct 1620 gaccagtaag gattggtatt gatatgccgt tggtggctgt gaacgcggcg atggacaatg 1680 gactataagt atttgcagta tacatatatc tttcacatca caagggggaa cagatatacg 1740 gactagactt ttggagtact gttggctcag tagtgtaatt gatgaggcgt cagtgaagcc 1800 cagtggtacc gcaaaaaaaa aaaaaaaaaa aa 1832 <210> 7 <211> 2010 <212> DNA <213> Artificial Sequence <220> <223> MnP cDNA sequence of Polyporellus brumalis <400> 7 acgcggggga cgtcccattc tactgaacag tcccactccc tcagcctgtc agcaacgtgt 60 cagcattcag gcgtaccaca aggaccaata tacgtacaac tgtcatgcaa atatattggc 120 tcgaagtgcg ctgcgaaata cgcgtgctcg gacgacactc ccaccaagaa cccaataccg 180 gtcctccgat ctgcccactg ctgtggtaag tctacccacc tgcatctgcc catctcatca 240 actaggtccc tgttcgtaga ttctctcgaa catccactcc actgtcgcat cgataccgtg 300 gtcgtgctcg cagggaccgg taccaatcgc gcaaccgctt cgaacagctc ctacgctgcc 360 cgcagccgag gttctgaacc acactaaggt ggctttagtt ctcaggagcg cacccgaagc 420 ttggttcagg tgcgatgcca gcagcgcgaa acatgacccg ctgggtaccc gccgtaccgt 480 ttgggtagtc attggccttc cattggctcc gtacatactg tacgccgaca caccgacgat 540 gcggaatgcc tcaaacaccc agggaatcgg gtgcctgcgt tcacgctgtg agaatgggtg 600 cagcgcgatt ggctggtctc cttgaacacg acctacgcct cgcaaggtcg agacatagca 660 tcaccgttaa ggctttggcg aacgagtata aaggagctgg ggcgacgacg accaaacctc 720 aggacatcta cctctcctca gtcgctccat ctcctcttcg gacaatggcc ttcaaaacac 780 tcgcgtcttt cgtctcagtt ctcgcggctt tccaagccgc taacggtgcc ctcgtccgcc 840 gagttacttg cgctgacggc agcgtcacgg cgaacgcggc gtgctgcgca ctcttcccgg 900 tgattcagga tctccagacg aacctcttcg acggcggcga gtgcggtgag gaggtccacg 960 aatcccttcg tctgaccttc cacgacgcca tcggtatatc ccccgctatc gcctcccgcg 1020 gccaattcgg tggtggcggc gccgacggtt cgatcgccat attcgacgac atcgagacta 1080 acttccacgc gaacaacggt gtcgatgaga tcatcggcga acaacgcccg ctcatccagc 1140 gccacaacat caccacggct gacttcatcc agctcgctgg tgccatcggt gtcagcaact 1200 gtcctggtgc tcctcgcctc gcggtgtttg tcggccgtcc tgatgccacc cagcccgcgc 1260 ccgacttgac cgtcccggag ccctttgaca ccgtcgacag catcctgcag cgcttcgacg 1320 atgccggtgg cttcactcct gaagaggttg tggctctcct tggctcccac actatcgccg 1380 cggccgacca cgtcgacccc tcgatccccg gaaccccctt cgactcgacg cctgagctct 1440 tcgacactca gttcttcatc gagacgcagc tccgtggtac gctcttcccc ggcactggcg 1500 gcaaccaggg tgaggtcgag tcgcccctcc gcggcgagct ccgtctccag tccgactctg 1560 agctcgctcg tgactctcgc actgcttgcg agtggcagtc cttcgtcaac aaccaggcca 1620 agctccagtc cgccttcgct gctgccttcc gcaagatgac gatcctcggc cacgacgagg 1680 gctcgctggt tgactgctcc gacgtcgtcc cgactccccc agccccggcc tcccaggcac 1740 acctccccgc cggtctcact cacaatgacg ttgagcaggc ttgtgccagc actcccttcc 1800 ccactctccc caccgacccc ggtccggcca cctccgttgc ccctgttcct ccctcctaaa 1860 gtgtcgcggt gctgctcaga ctggagatca ttcttcgtta tctcgggatg gctgctacat 1920 agatatgtaa ctcttaacca ctggtctatc attgagatta tgtgacaaca accgattcgt 1980 cccataaaaa aaaaaaaaaa aaaaaaaaaa 2010 <110> Korea (Korea Forest Research Institute) <120> Transformant expressing two kinds of ligninases, method for          preparing the transformant and method for degrading endocrine          disruptor by using the transformant <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Foward primer Sma I linker of Polyporellus brumalis <400> 1 cccgggatgg ccttcaaaac a 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer EcoR I linker of Polyporellus brumalis <400> 2 gaattcttag gagggaggaa cagg 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Foward primer Xho I linker containing Merulius tremellosus          promoter <400> 3 ctcgaggcac gctgttactg cc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer Xho I linker of Merulius tremellosus promoter <400> 4 ctcgagggcg tcagtgaagc c 21 <210> 5 <211> 3153 <212> DNA <213> Artificial Sequence <220> <223> gDNA sequence containing Merulius tremellosus promoter <400> 5 gcacgctgtt actgcctgcg tacgcagccc agtgcaacca tcgcctaaca tctgacagtc 60 tatcagttgc aggacaaccc ttagcgatcg ctttgatgtc tagcgaagta cgaggaaagt 120 cagatgctgt tctatctcct acgcagatgc ggacgagatc cagtggaacg ggtatggctc 180 ccccattccc agcctgttct cagctcacgt cttcgccggt cggagtgggt ctcatctgta 240 agtaagtttt tctatcaaac ttagcccgtc gtctcttatt gaactatcac tagcaaaccc 300 ttctccagcg cgagcaagat ccagacactg gttgctcgtg ggagtcactc ttcagtcgcc 360 ttcagtttgg gtccgtgtgt acgtctgatg gctgccggca ccgaaatttg agacaaggtg 420 ggtcgagctt cacccgatga cacggcctgc gattttgaca ttcaagctta tgttcctgac 480 aggtcatctc acgcccaggt ctgcgctcac gcgatattcg gcaaactcta cagaaaatat 540 acattttctg cgaaggccac gagcggcagg ccatcgggat tgccggacat gaaccagatt 600 ctgtaccgag gtgtacggcc ttgtatatct gacttgctct atttgttgtg gtcagaggta 660 tattaaccag tgagtctcct gcctgtgctc cccagttccc cgctgacaag ggattaccag 720 agggcctctt caccaatgtt ttccttcact cgttctactg cgctcgtcgt cgcaggcctt 780 tccgctggtg ccatcgctgc catcggccct gtgactgact ttcatatcgt caatgcagcc 840 atcgcccccg acggtatctc tcgtcaggct gtgcttgctg agggcacatt ccctggtcct 900 cttattactg gcaacaaggt aggaccaact ccgtgttgtc ttttgttcat ttattcatca 960 tcgtaccgaa aataacaggg cgacaacttt caaatcaatg ttatcgacga gctgaccaat 1020 gctaccatgc tcaagtcgac gacgattgtt agttcacgca atactctatt ccacgctctc 1080 tggtgcttac tcttcacttc agcactggca tggattcttc cagcacggaa cgaactgggc 1140 tgatggtccg gccttcgtca atcaatgtcc tattgctacc gggaattctt tcttgtacaa 1200 cttcaacgca cccgatcaag ccggtttgtt gttttacaag gcttcacgta cattgcgtac 1260 tcacgtggtg atgtcggccc tcaggtacat tctggtatca tagtcatctg tccactcagt 1320 actgtgacgg tttgcgtggt ccttttgtgg tctacgatcc caatgatcct tatgcggatt 1380 tgtacgacgt tgatgacggt aagcattcag ccataccgaa gtaacgtagg ctgatatctg 1440 cgccagattc taccgtcatc acgctggccg actggtatca tacggctgcc cgcctcggcg 1500 gtcctttccc gtaagtttaa acagttcatc gcagacagtc aacgacgtat cgactgacct 1560 ttatctaagc aatgccgaca ctactttgat taatggccta ggcagatgcg gtgaaccggg 1620 cgctcctgtc tctgatctcg ctgttatcaa cgtcgaggct ggcaagcggt atgtccacga 1680 tttcagccat gccattcccc tttgtatgct catttcggtc atttgtatag ttatcgaatg 1740 cgcttggttt cgatctcctg tgactcgtgg ttcaatttca ccatcgatgg acacgatatg 1800 accataattg aagttgatgg cgtcaaccac gagacgttga ccgtagacca aatccagatc 1860 ttcgccgctc agcgttactc tttcatcgta agcattcatt gtttctcgta catggagtca 1920 gactctgatc atgaatatat tatagttgga agcaaaccag cctgtcgata actactggat 1980 tcgcgcaaac ccgggtaccg gcatcactca agggttcgag ggcggcatca actcggccat 2040 tctgcgctat gctggcgctg acgaggttga acctaccact gcgttagttg cgagcacctc 2100 tccgttggcc gagggagatt tgcaccctct agacaaccct gctgccgtat gttcatgtcg 2160 catgtccatt cacaaccgat actgatcgat gctctaccag cctggccaac ccttcccagg 2220 cggcgtcgat tacgctttga atttgaactt gacattcgtc agtctagaaa gtcgtctgtg 2280 tgaaaccaca tctgatgtgc tttgcaggat ggaccgaccc ttaagttcct gatcaatggg 2340 gtgtctttcg tacccccgac agttccggtg ctcctgcaga ttctcagtgg tgcgcagacc 2400 gcacaagatc ttcttcctac tgggagtgtc tactccttgc cctcgaacgc gaccattgaa 2460 ttgagtctcc ctgctggtcc tgcaggcgga cctcatccct tccacttgca cggcgtgagt 2520 ttgcctattc ctaatttgca tatggtatta acttaacctt catatagcac acgttcagtg 2580 tggttcaagg tgcaggacaa acggtgccga actacgtgaa tcctgtccgc cgagatgttg 2640 tcagcatcgg agctgttccc gacaatgtca ccattcgttt cgaggtacga atgcaatcag 2700 tatagatgag agggctcttt ttgaccaaag gtcatcgtta cagaccaaca atcccggccc 2760 atggttcctc cattgccaca tcgactggca cttggatgct ggcttcgccg aggtattcgc 2820 tgaagacatc cctgatatcg cttcggtcaa ccctgtcctt agtgagtcac acacatcatg 2880 cacgggtatc aatcaccatg tactgatgac tgccttagcc gactggtcca atctgtgccc 2940 aatctacgac gctctcgatc cttctgacca gtaaggattg gtattgatat gccgttggtg 3000 gctgtgaacg cggcgatgga caatggacta taagtatttg cagtatacat atatctttca 3060 catcacaagg gggaacagat atacggacta gacttttgga gtactgttgg ctcagtagtg 3120 tagttgatga ggcgtcagtg aagccggatc cac 3153 <210> 6 <211> 1832 <212> DNA <213> Artificial Sequence <220> <223> Laccase cDNA sequence of Merulius tremellosus <400> 6 gggggatagg gggggaggaa gcggagttcc ccgctgacaa ggggttgcca gagggcctct 60 tcaccaatgt tttccttcgc tcgttctact gcgctcgtcg tcgcaggcct tttcgttggt 120 gccatcgccg ccatcggccc tgtgactgac tttcatatcg tcaatgcagc catcgccccc 180 gacggtatct ctcgtcaggc cgtgcttgct gagggcacat tccctggtcc tctcattact 240 ggcaacaagg gcgacaactt ccaaatcaat gttatcgacg agctaaccaa tgctaccatg 300 ctcaagtcga cgacgatcca ctggcatgga ttcttccagc acggaacgaa ctgggctgat 360 ggtccggcct tcgtcaatca atgtcctatt gctaccggga attctttctt gtacaacttc 420 aacgcacccg atcaagccgg tacattctgg tatcatagtc atctgtccac tcagtactgt 480 gacggtttgc gtggtccttt tgtggtctac gatcccaatg atccttatgc ggatttgtac 540 gacgttgatg acgattctac cgtcatcacg ctggccgact ggtatcatac ggctgcccgc 600 ctcggcggtc ctttccccaa tgccgacact actttgatta atggcctggg cagatgcggt 660 gaaccgggcg ctcctgtctc tgatctcgct gttatcaacg tcgaggctgg caagcgttat 720 cgaatgcgct tggtttcgat ctcctgtgac tcgtggttca atttcaccat cgatggacac 780 gatatgacca taattgaagt tgatggcgtc aaccacgaga cgttgaccgt agaccaaatc 840 cagatcttcg ccgctcagcg ttactctttc atcttggaag caaaccagcc tgtcgataac 900 tactggattc gcgcaaaccc gggtaccggc atcactcaag ggttcgaggg cggcatcaac 960 tcggccattc tgcgctatgc tggcgctgac gaggttgaac ctaccactgc gttagttgcg 1020 agcacctctc cgttggccga gggagatttg caccctctag acaaccctgc tgccnctggc 1080 caacccttcc caggcggcgt cgattacgct ttgaatttga acttgacatt cgatggaccc 1140 acccttaagt tcctgatcaa tggggtgtct ttcgtacccc cgacagttcc ggtgctcctg 1200 cagattctca gtggtgcgca gaccgcacaa gatcttcttc ctactgggag tgtctactcc 1260 ttgccctcga acgcgaccat tgaattgagt ctccctgctg gtcctgcagg cggacctcat 1320 cccttccact tgcacggcca cacgttcagt gtggttcaag gtgcaggaca aacggtgccg 1380 aactacgtga atcctgtccg ccgagatgtt gtcagcatcg gagctgttcc cgacaatgtc 1440 accattcgtt tcgagaccaa caatcccggc ccatggttcc tccattgcca catcgactgg 1500 cacttggatg ctggcttcgc cgtggtattc gctgaagaca tccccgatat cgcttcggtc 1560 agccctgtcc ctaccgactg gtccaatctg tgcccaatct acgacgctct cgatccttct 1620 gaccagtaag gattggtatt gatatgccgt tggtggctgt gaacgcggcg atggacaatg 1680 gactataagt atttgcagta tacatatatc tttcacatca caagggggaa cagatatacg 1740 gactagactt ttggagtact gttggctcag tagtgtaatt gatgaggcgt cagtgaagcc 1800 cagtggtacc gcaaaaaaaa aaaaaaaaaa aa 1832 <210> 7 <211> 2010 <212> DNA <213> Artificial Sequence <220> <223> MnP cDNA sequence of Polyporellus brumalis <400> 7 acgcggggga cgtcccattc tactgaacag tcccactccc tcagcctgtc agcaacgtgt 60 cagcattcag gcgtaccaca aggaccaata tacgtacaac tgtcatgcaa atatattggc 120 tcgaagtgcg ctgcgaaata cgcgtgctcg gacgacactc ccaccaagaa cccaataccg 180 gtcctccgat ctgcccactg ctgtggtaag tctacccacc tgcatctgcc catctcatca 240 actaggtccc tgttcgtaga ttctctcgaa catccactcc actgtcgcat cgataccgtg 300 gtcgtgctcg cagggaccgg taccaatcgc gcaaccgctt cgaacagctc ctacgctgcc 360 cgcagccgag gttctgaacc acactaaggt ggctttagtt ctcaggagcg cacccgaagc 420 ttggttcagg tgcgatgcca gcagcgcgaa acatgacccg ctgggtaccc gccgtaccgt 480 ttgggtagtc attggccttc cattggctcc gtacatactg tacgccgaca caccgacgat 540 gcggaatgcc tcaaacaccc agggaatcgg gtgcctgcgt tcacgctgtg agaatgggtg 600 cagcgcgatt ggctggtctc cttgaacacg acctacgcct cgcaaggtcg agacatagca 660 tcaccgttaa ggctttggcg aacgagtata aaggagctgg ggcgacgacg accaaacctc 720 aggacatcta cctctcctca gtcgctccat ctcctcttcg gacaatggcc ttcaaaacac 780 tcgcgtcttt cgtctcagtt ctcgcggctt tccaagccgc taacggtgcc ctcgtccgcc 840 gagttacttg cgctgacggc agcgtcacgg cgaacgcggc gtgctgcgca ctcttcccgg 900 tgattcagga tctccagacg aacctcttcg acggcggcga gtgcggtgag gaggtccacg 960 aatcccttcg tctgaccttc cacgacgcca tcggtatatc ccccgctatc gcctcccgcg 1020 gccaattcgg tggtggcggc gccgacggtt cgatcgccat attcgacgac atcgagacta 1080 acttccacgc gaacaacggt gtcgatgaga tcatcggcga acaacgcccg ctcatccagc 1140 gccacaacat caccacggct gacttcatcc agctcgctgg tgccatcggt gtcagcaact 1200 gtcctggtgc tcctcgcctc gcggtgtttg tcggccgtcc tgatgccacc cagcccgcgc 1260 ccgacttgac cgtcccggag ccctttgaca ccgtcgacag catcctgcag cgcttcgacg 1320 atgccggtgg cttcactcct gaagaggttg tggctctcct tggctcccac actatcgccg 1380 cggccgacca cgtcgacccc tcgatccccg gaaccccctt cgactcgacg cctgagctct 1440 tcgacactca gttcttcatc gagacgcagc tccgtggtac gctcttcccc ggcactggcg 1500 gcaaccaggg tgaggtcgag tcgcccctcc gcggcgagct ccgtctccag tccgactctg 1560 agctcgctcg tgactctcgc actgcttgcg agtggcagtc cttcgtcaac aaccaggcca 1620 agctccagtc cgccttcgct gctgccttcc gcaagatgac gatcctcggc cacgacgagg 1680 gctcgctggt tgactgctcc gacgtcgtcc cgactccccc agccccggcc tcccaggcac 1740 acctccccgc cggtctcact cacaatgacg ttgagcaggc ttgtgccagc actcccttcc 1800 ccactctccc caccgacccc ggtccggcca cctccgttgc ccctgttcct ccctcctaaa 1860 gtgtcgcggt gctgctcaga ctggagatca ttcttcgtta tctcgggatg gctgctacat 1920 agatatgtaa ctcttaacca ctggtctatc attgagatta tgtgacaaca accgattcgt 1980 cccataaaaa aaaaaaaaaa aaaaaaaaaa 2010  

Claims (14)

(i) 락카아제(laccase) 유전자와 망간 퍼옥시다아제(Mn peroxidase) 유전자를 동시에 발현시키는 것을 특징으로 하고, (ii) 상기 락카아제의 발현유전자는 서열번호 6으로 표시되는 염기서열인 것을 특징으로 하고, (iii) 상기 망간 퍼옥시다아제의 발현 유전자는 서열번호 7로 표시되는 염기서열인 것을 특징으로 하는 재조합벡터 pMnP-gLac.(i) simultaneously expressing the laccase gene and the manganese peroxidase gene, and (ii) the expression gene of the laccase is characterized in that the nucleotide sequence represented by SEQ ID NO: 6 , (iii) The expression vector of the manganese peroxidase is a recombinant vector pMnP-gLac, characterized in that the nucleotide sequence represented by SEQ ID NO: 7. 삭제delete (i) 락카아제(laccase) 유전자와 망간 퍼옥시다아제(Mn peroxidase) 유전자를 동시에 발현시키는 것을 특징으로 하고, (ii) 도 16으로 표시되는 개열지도를 갖는 것을 특징으로 하는 재조합 벡터 pMnP-gLac.(i) expressing a laccase gene and a manganese peroxidase gene at the same time, and (ii) a recombinant vector pMnP-gLac characterized by having a cleavage map shown in FIG. 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete
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