KR100888481B1 - Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof - Google Patents

Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof Download PDF

Info

Publication number
KR100888481B1
KR100888481B1 KR1020070082453A KR20070082453A KR100888481B1 KR 100888481 B1 KR100888481 B1 KR 100888481B1 KR 1020070082453 A KR1020070082453 A KR 1020070082453A KR 20070082453 A KR20070082453 A KR 20070082453A KR 100888481 B1 KR100888481 B1 KR 100888481B1
Authority
KR
South Korea
Prior art keywords
ala
leu
pro
gly
asp
Prior art date
Application number
KR1020070082453A
Other languages
Korean (ko)
Other versions
KR20090017925A (en
Inventor
김명길
유선화
Original Assignee
대한민국
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 대한민국 filed Critical 대한민국
Priority to KR1020070082453A priority Critical patent/KR100888481B1/en
Publication of KR20090017925A publication Critical patent/KR20090017925A/en
Application granted granted Critical
Publication of KR100888481B1 publication Critical patent/KR100888481B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01003—Fatty-acid peroxidase (1.11.1.3)

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

본 발명은 겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자 및 이를 함유하는 재조합 벡터에 관한 것으로, 본 발명은 플라스틱 가소제로 사용하는 프탈레이트류의 생물학적 분해에 이용되는 백색 부후균, 겨울우산버섯의 리그닌 분해 관여 효소인 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자의 유전적 기능을 규명함으로써, 백색 부후균을 포함한 진균류의 목질 분해의 유전적 메카니즘을 이해할 수 있게 한다. 또한, 본 발명은 상기 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자에 의한 리그닌 분해 기능성이 강화된 식용버섯을 포함한 진균류의 생산 및 이용에 기여할 수 있다.The present invention relates to a manganese peroxidase gene of a winter umbrella, and a recombinant vector containing the same. The present invention relates to enzymes involved in lignin degradation of white fungi and winter umbrellas, which are used for the biological decomposition of phthalates used as plasticizers. By identifying the genetic function of the manganese peroxidase gene, it is possible to understand the genetic mechanism of wood degradation of fungi, including white fungi. In addition, the present invention can contribute to the production and use of fungi, including edible mushrooms with enhanced lignin degradation by the manganese peroxidase gene.

겨울우산버섯, 망간에이즈 퍼옥시다아제 Winter Umbrella, Manganese AIDS Peroxidase

Description

겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자 및 이를 함유하는 재조합 벡터{Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof}Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing lamb}

본 발명은 겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자 및 이를 함유하는 재조합 벡터에 관한 것으로, 더욱 상세하게는 플라스틱 가소제로 사용하는 프탈레이트류의 생물학적 분해에 이용된 백색 부후균인 겨울우산버섯(Polyporus brumalis)의 리그닌 분해 효소 유전자의 분리 및 발현 특성을 조사하고 형질전환 기술을 확립하고, 이를 이용하여 환경호르몬을 포함한 목질 내 고분자 물질의 분해 기능이 확대된 백색부후균의 형질전환체를 얻는 것에 관한 것이다.The present invention relates to a manganese peroxidase gene of a winter umbrella, and a recombinant vector containing the same. More specifically, a winter fungi ( Polyporus brumalis) , which is a white fungus used for the biological degradation of phthalates, which are used as plastic plasticizers. To investigate the isolation and expression characteristics of the lignin degrading enzyme gene and to establish the transformation technology, and to obtain a transformant of the white fungus expanded the degradation function of the polymer material in the wood, including environmental hormones.

내분비계 장애물질(Endocrine Disruptor)은 환경 중 배출된 화학물질이 체내에 유입되어 마치 호르몬처럼 작용하여 생태계 및 인간의 생식기능 저하, 기형, 성장장애, 암 등을 유발하여 오존층 파괴, 지구 온난화 문제와 함께 세계 3대 환경문제로 등장하였다. 내분비계 장애물질은 일반적으로 물질의 종류에 따라 저해호르몬 의 종류 및 저해방법이 각각 다르다.Endocrine Disruptors are chemicals released from the environment that act like hormones, causing reproductive harm, deformity, growth disorders, and cancers in the ecosystem and humans. Together, they emerged as one of the world's three major environmental problems. Endocrine disruptors generally differ in the type of inhibitor and the method of inhibition, depending on the type of substance.

환경호르몬으로 작용하여 인체에 영향을 미치는 프탈레이트류는 백색부후균인 겨울우산버섯(Polyporus brumalis)에 의해 분해될 수 있다. 리그닌분해효소인 망간에이즈 퍼옥시다아제(Manganese peroxidase)는 대부분 백색부후균에서 발견되고 있다. 이러한 장점으로 인해 리그닌을 포함하여 여러 방향족 고분자 화합물을 분해시켜 저분자화 한다.Phthalates, which act as environmental hormones and affect the human body, can be decomposed by the white rot fungi, Polyporus brumalis . Manganese peroxidase, a lignin degrading enzyme, is found in most white fungi. Due to these advantages, many aromatic polymer compounds, including lignin, are degraded to lower their molecular weight.

그러나 리그닌분해효소의 유전자적 특성에 관해서는 널이 알려진 바 없다. 특히 목질 내의 리그닌 분해력이 우수한 것으로 알려진 판막버섯(Phanerochate chrysosporium), 구름버섯(Trametes versicolor)의 리그닌 분해효소 라카아제, Mn-peroxidase(mnp1-mnp3), Lignin-peroxidase(lipA -lipJ, LPGI-LPGIV) 유전자를 이용하여 Aspergillus nidulans로부터 분리한 α-amylase 프로모터 조절에 의해 열적으로 안정하고 생물학적 복구법에 이용될 수 있도록 그 기능을 강화한 형질 전환 기술은 활발히 진행되고 있다.However, nothing is known about the genetic properties of ligase. In particular, Phanerochate chrysosporium , Trametes versicolor lignin degrading enzyme laccase, Mn-peroxidase ( mnp1-mnp3 ), Transformation technology that is thermally stable and regulated by the α-amylase promoter isolated from Aspergillus nidulans using Lignin-peroxidase ( lipA - lipJ , LPGI-LPGIV ) genes can be used for biological repair. It is becoming.

리그닌 분해효소를 사용할 경우에는 소량의 화합물로 적은 에너지를 사용하여 고효율의 리그닌을 처리할 수 있고 환경오염물의 방출도 줄일 수 있는 장점을 가지고 있다. 또한, 리그닌 분해효소는 다양한 종류의 자연계에 존재하며 인체에 유해하거나 환경에 해를 끼치는 방향족 화합물을 분해할 수 있어 환경보전의 측면에서 매우 중요한 관심을 끌고 있다(Field, J. F., et al., Trend in Biotechnol. 1993, 11, 44-49).When lignin degrading enzyme is used, it is possible to treat efficiencies of lignin with a small amount of compounds with little energy and to reduce the emission of environmental pollutants. In addition, lignin degrading enzymes exist in various kinds of natural systems and can decompose aromatic compounds that are harmful to humans or harmful to the environment, and thus are of great interest in environmental conservation (Field, JF, et al., Trend). in Biotechnol. 1993, 11, 44-49).

백색부후균의 경우는 각종 화합물에 대하여 비특이적으로 작용할 수 있는 기 작을 보유하여 다양한 난분해성 화합물을 분해할 수 있으며, 분해과정이 세포외에서 이루어지기 때문에 고농도의 독성화합물과 불용성 화합물의 처리가 가능하고 현장적용에 있어서도 생장조건의 특이성으로 선택적인 성장을 보이므로 다른 미생물의 영향을 배제할 수 있는 장점이 있다. 이러한 특징 때문에 백색부후균은 생물학적 환경정화에 유용하게 사용될 수 있으며, 이를 적용하기 위해서는 우수한 균주를 선별하는 기술, 백색부후균의 균사체를 대량으로 배양하는 기술, 리그닌분해효소를 효소활성이 높게 생산하는 기술 등이 필요하다.In case of white fungus, it can decompose various hardly decomposable compounds by having a mechanism that can act nonspecifically on various compounds, and it is possible to treat high concentration of toxic and insoluble compounds because the decomposition process is carried out extracellularly. In the application, since it shows selective growth due to the specificity of growth conditions, there is an advantage that can exclude the influence of other microorganisms. Because of these characteristics, white fungus can be usefully used for biological environment purification, and to apply this, a technique for selecting excellent strains, a technique for cultivating mycelia of white fungus in large quantities, and producing lignin degrading enzyme with high enzymatic activity Skills are needed.

본 발명의 목적은 플라스틱 가소제로 사용하는 프탈레이트류의 생물학적 분해에 이용되는 백색 부후균인 겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자 및 이를 포함하는 재조합 벡터를 제공하는데 있다. Disclosure of Invention An object of the present invention is to provide a manganese peroxidase gene of winter umbrella, which is a white fungus used for the biological degradation of phthalates used as plastic plasticizers, and a recombinant vector comprising the same.

본 발명의 다른 목적은 서열 7, 서열 8, 서열 9, 서열 10, 서열 11 및 서열 12 중에서 선택된 어느 하나의 아미노산 서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 효소를 제공하는데 있다. Another object of the present invention to provide a manganese peroxidase ( Polyporus brumalis ) manganese peroxidase enzyme consisting of any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 It is.

본 발명의 다른 목적은 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터를 함유하는 형질전환체를 제공하는데 있다. Another object of the present invention is to provide a transformant containing a recombinant vector containing a manganese peroxidase gene.

본 발명의 다른 목적은 망간에이즈 퍼옥시다아제 유전자를 백색부후균에 도 입하여 환경호르몬을 포함한 목질 내 고분자 물질의 분해 기능이 확대된 백색부후균의 형질전환체를 제공하는데 있다.Another object of the present invention is to introduce a manganese peroxidase gene to white fungus to provide a transformant of the white fungus expanded the degradation function of the polymer material in the wood, including environmental hormones.

상기한 목적을 달성하기 위한 본 발명은 서열 1, 서열 2, 서열 3, 서열 4, 서열 5 및 서열 6 중에서 선택된 어느 하나의 염기서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자를 제공한다. The present invention for achieving the above object is Polyurus brumalis manganese peroxidase consisting of any one base sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 6 ) Provides genes.

본 발명은 상기 유전자가 코딩하는 서열 7, 서열 8, 서열 9, 서열 10, 서열 11 및 서열 12 중에서 선택된 어느 하나의 아미노산서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 효소를 제공한다. The present invention is a manganese peroxidase ( Polyporus brumalis ) manganese peroxidase enzyme consisting of any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 encoded by the gene. To provide.

본 발명은 상기 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터를 제공한다. The present invention provides a recombinant vector containing the manganese peroxidase gene.

본 발명은 상기 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터로 형질전환된 대장균(E. coli)을 제공한다.The present invention provides E. coli transformed with a recombinant vector containing the manganese peroxidase gene.

본 발명은 상기 망간에이즈 퍼옥시다아제 유전자를 함유하는 리그닌 분해 기능성이 강화된 겨울우산버섯을 제공한다.The present invention provides a winter umbrella mushroom with enhanced lignin degradation function containing the manganese peroxidase gene.

본 발명은 플라스틱 가소제로 사용하는 프탈레이트류의 생물학적 분해에 이용되는 백색 부후균인 겨울우산버섯의 리그닌 분해 관여 효소인 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자의 유전적 기능을 규명함으로써, 백색 부후균을 포함한 진균류의 목질 분해의 유전적 메카니즘을 이해할 수 있게 한다. 또한, 본 발명은 상기 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자에 의한 리그닌 분해 기능성이 강화된 식용버섯을 포함한 진균류의 생산 및 이용에 기여할 수 있다.The present invention provides a white fungus by identifying the genetic function of the manganese peroxidase gene, an enzyme involved in the lignin digestion of winter umbrella, which is a white fungus used for the biological degradation of phthalates used as plastic plasticizers. To understand the genetic mechanism of wood degradation of fungi, including In addition, the present invention can contribute to the production and use of fungi, including edible mushrooms with enhanced lignin degradation by the manganese peroxidase gene.

본 발명은 서열 1, 서열 2, 서열 3, 서열 4, 서열 5 및 서열 6 중에서 선택된 어느 하나의 염기서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자를 나타낸다.The present invention SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, the sequence consisting of any one of nucleotide sequence selected from SEQ ID NO: 5 and 6 umbrella winter mushroom (Polyporus brumalis ) represents a manganese peroxidase gene.

본 발명은 상기에서 언급한 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터를 포함한다.The present invention includes recombinant vectors containing the manganese peroxidase gene mentioned above.

본 발명은 상기에서 언급한 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터로 형질전환된 대장균(E. coli)을 포함한다.The present invention includes E. coli transformed with a recombinant vector containing the aforementioned manganese peroxidase gene.

본 발명은 상기에서 언급한 망간에이즈 퍼옥시다아제 유전자를 함유하는 리그닌 분해 기능성이 강화된 겨울우산버섯을 포함한다.The present invention includes a winter umbrella mushroom with enhanced lignin-degrading functionality containing the manganese peroxidase gene mentioned above.

본 발명은 서열 7, 서열 8, 서열 9, 서열 10, 서열 11 및 서열 12 중에서 선택된 어느 하나의 아미노산 서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망 간에이즈 퍼옥시다아제(manganese peroxidase) 효소를 포함한다.The present invention is a winter umbrella consisting of any one amino acid sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 12 ( Polyporus) brumalis ) manganese peroxidase enzyme.

본 발명을 상세히 설명하면 다음과 같다.The present invention is described in detail as follows.

본 발명을 위하여 망간에이즈 퍼옥시다아제(manganese peroxidase: MnP) 분해 효소에 의해 자연계에 존재하는 방향족 화합물을 분해 가능한 여러 백색부후균을 이용하여 플라스틱 가소제인 프탈레이트류에 대한 저항성 및 분해능을 조사한 결과 겨울우산버섯(Polyporus brumalis)이 우수한 분해능을 보였고 그 분해산물의 에스트로겐성 저감효과도 뛰어난 것으로 나타났다. 이러한 백색부후균의 리그닌 분해력과 환경 정화의 기능이 강화된 백색 부후균 리그닌 분해 효소의 유전자적 기능을 구명하는 것이 필요하였고 이를 바탕으로 백색 부후균의 리그닌 분해 효소 유전자를 분리하고 기능성이 강화된 형질전환 백색 부후균을 얻기 위하여 리그닌 분해 효소 유전자의 발현 유도 및 검정을 하였다.Winter Umbrella mushrooms were tested for resistance and degradability of phthalates, plastic plasticizers, using various white fungi capable of degrading aromatic compounds present in nature by manganese peroxidase (MnP) degrading enzymes. ( Polyporus brumalis ) showed excellent resolution, and the degradation effect of the product was also excellent. It was necessary to investigate the genetic function of the white rot lignin degrading enzyme which enhanced the lignin degrading power and environmental purification function of the white rot fungus. Expression of the lignin degrading enzyme gene was induced and assayed to obtain converted white fungi.

본 발명은 환경호르몬에 오염된 토양, 수질 및 생물분해 반응기에서의 생물학적 분해 및 복구법에 이용되는 백색부후균인 겨울우산버섯(Polyporus brumalis)의 리그닌 분해효소 유전자를 분리하고 발현 특성을 조사하였다. 이를 위하여 겨울우산버섯(Polyporus brumalis)의 리그닌 분해효소 중 망간에이즈 퍼옥시다아제(manganese peroxidase: MnP) 유전자를 분리하여 유전자(full gene)를 확보하였고 환경호르몬 분해 시 나타나는 발현 특성을 조사하였다. In the present invention, the lignin degrading enzyme gene of Polyporus brumalis , a white rot bacterium used for biological degradation and recovery in soil, water quality and biodegradation reactor contaminated with environmental hormones, was isolated and examined for expression characteristics. To this end, winter mushroom umbrella lignin decomposition AIDS manganese peroxidase enzyme in the (Polyporus brumalis): to remove the (manganese peroxidase MnP) gene was obtained a gene (full gene) was investigated expression characteristics that appear when the environmental hormone decomposing.

본 발명은 겨울우산버섯의 망간에이즈 퍼옥시다아제(manganese peroxidase)의 유전자(full gene)를 확보하기 위하여 겨울우산버섯을 SSC 배지에서 배양한 후 회수하여 갈고 트리졸(Trizol: invitrogen)을 사용하여 전체 RNA(total RNA)를 분리할 수 있다. In order to secure the full gene of manganese peroxidase of winter umbrella mushrooms, the present invention is recovered after incubating the winter umbrella mushrooms in SSC medium, and then using a trizol (Trizol: invitrogen). (total RNA) can be isolated.

상기 total RNA로부터 cDNA의 합성은 total RNA를 주형(template)으로 하고, cDNA를 합성 키트로 Cap Fishing Full-length cDNA Premix Kit (Seegene) 또는 BD Rapid cDNA Amplified Ends (RACE) kit (Clontech)을 사용하여 cDNA를 합성할 수 있다.Synthesis of cDNA from the total RNA using total RNA as a template, and using cDNA as a synthesis kit, Cap Fishing Full-length cDNA Premix Kit (Seegene) or BD Rapid cDNA Amplified Ends (RACE) kit (Clontech) cDNA can be synthesized.

상기 cDNA로부터 망간에이즈 퍼옥시다아제(manganese peroxidase)의 PCR 산물을 얻을 수 있으며, 상기 PCR 산물을 pGEM-T Vector system1 kit 또는 TOPO-TA kit를 사용하여 벡터(vector)에 클로닝 하는 것이 바람직하다.A PCR product of manganese peroxidase can be obtained from the cDNA, and the PCR product is preferably cloned into a vector using a pGEM-T Vector system1 kit or a TOPO-TA kit.

본 발명은 겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자의 염기서열을 분석하여 Pbmnp1 (서열 1), Pbmnp2 (서열 2), Pbmnp3 (서열 3), Pbmnp4 (서열 4), Pbmnp5 (서열 5) 및 Pbmnp6 (서열 6)로 명명하였다. The invention Pbmnp1 (SEQ ID NO: 1), Pbmnp2 (SEQ ID NO: 2), Pbmnp3 (SEQ ID NO: 3), Pbmnp4 (SEQ ID NO: 4), Pbmnp5 (SEQ ID NO: 5) by analyzing the nucleotide sequence of the AIDS manganese peroxidase gene from winter mushroom umbrella And Pbmnp6 (SEQ ID NO: 6).

본 발명은 상기 망간에이즈 퍼옥시다아제 유전자 중에서 선택된 어느 하나를 함유하는 재조합 벡터를 제조할 수 있다. The present invention can produce a recombinant vector containing any one selected from the manganese peroxidase gene.

본 발명은 상기 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터를 함유하는 형질전환체를 제조할 수 있다. The present invention can produce a transformant containing a recombinant vector containing the manganese peroxidase gene.

또한, 본 발명은 상기 형질전환체를 이용하여 망간에이즈 퍼옥시다아제 유전자를 함유하는 리그닌 분해 기능성이 강화된 겨울우산버섯을 제조할 수 있다. In addition, the present invention can be prepared for the winter umbrella mushrooms with enhanced lignin degradation function containing the manganese peroxidase gene using the transformant.

환경호르몬으로 작용하여 인체에 영향을 미치는 프탈레이트류가 백색부후균인 겨울우산버섯(Polyporus brumalis)에 의해 분해될 수 있으며, 난분해성 위해물 질이 가지는 특징 중의 하나인 에스트로겐성을 저감시킨다.Phthalates, which act as environmental hormones and affect the human body, can be decomposed by the white rot fungi, Polyporus brumalis , and reduce estrogen, one of the characteristics of hardly degradable hazards.

따라서 본 발명은 플라스틱 가소제로 사용하는 프탈레이트류의 생물학적 분해에 이용되는 백색 부후균인 겨울우산버섯의 망간에이즈 퍼옥시다아제 유전자를 제공하고, 본 발명은 백색 부후균의 리그닌 분해 관여 효소의 유전적 기능을 구명함으로써 백색 부후균을 포함한 진균류의 목질 분해의 유전적 메카니즘을 이해하여 선발 유전자에 의한 기능성이 강화된 식용버섯을 포함한 진균류의 생산 및 이용에 기여할 수 있다고 사료된다.Accordingly, the present invention provides a manganese peroxidase gene of winter umbrella mushroom, which is a white fungus used for the biological degradation of phthalates used as plastic plasticizers, and the present invention provides a genetic function of the enzyme involved in lignin degradation of white fungi. By understanding the genetic mechanism of wood degradation of fungi, including white fungi, it is thought to contribute to the production and use of fungi, including edible mushrooms with enhanced functionality by selection genes.

이하 본 발명을 다음 실시 예에 의하여 더욱 구체적으로 설명하고자 한다. 다만, 이들 실시예는 본 발명을 더욱 상세하게 설명하기 위해 제안된 것으로서 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are proposed to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

<실시예 1> 균주 및 배양Example 1 Strains and Cultures

백색부후균의 리그닌분해효소 유전자를 분리하기 위하여 공시균주로는 겨울우산버섯(Polyporus brumalis)으로 하고, 리그닌분해효소 활성을 증가시키기 위해 본래의 박층고정상(SSC;Shallow stationary culture) 배지를 약간 변화시켜 사용하였다. In order to isolate the leucine enzyme gene of the white rot bacterium, the test strain was Polyurus brumalis , and slightly changed the original SSC (Shallow stationary culture) medium to increase the ligase activity. Used.

본 발명에서 사용된 액체배지 1L에 글루코오스(glucose) 10g, (NH4)2C4H4O6 0.5g, 티아민(Thiamine)-HCl 0.5㎎, KH2PO4 0.5g, MgSO4·7H2O 0.5g, Ca(H2PO4)2 0.5g, 그리고 소량의 무기질 혼합물(MgSO4 3g, NaCl 1g, MnSO4 0.5g, FeSO4·7H2O 0.1g, CoCl2 0.1g, ZnSO4·7H2O 0.1g, CuSO4 0.1g, AlK(SO4)2·12H2O 10㎎, 니트릴로아세틱에시드(nitriloacetic acid) 1.5g을 1L의 증류수에 (0.2㎛ 여과지를 이용하여 여과 후 배지에 첨가) 첨가하였으며, pH는 4.5로 조정하였다. 접종은 PDA 배지에서 7일간 선 배양된 균을 대상으로 8㎜ 직경의 플러그(plug)를 제조한 후 배지 20㎖ 당 1 개의 플러그(plug)를 접종하였으며 25℃에서 10일간 진탕배양을 수행하였다.10 g of glucose (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 in 1 L of the liquid medium used in the present invention 0.5 g of O, 0.5 g of Ca (H 2 PO 4 ) 2 , and a small amount of mineral mixture (MgSO 4 3g, NaCl 1g, MnSO 4 0.5g, FeSO 4 7H 2 O 0.1g, CoCl 2 0.1g, ZnSO 4 0.1g of 7H 2 O, 0.1g of CuSO 4 , 10mg of AlK (SO 4 ) 2 · 12H 2 O, 1.5g of nitriloacetic acid (nitriloacetic acid) in 1L of distilled water The pH was adjusted to 4.5. The inoculation was performed for 7 days of precultured bacteria in PDA medium, and 8 mm diameter plugs were prepared. Inoculation was carried out for 10 days shaking culture at 25 ℃.

<실시예 2> 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자 분리Example 2 Manganese Peroxidase Gene Isolation

겨울우산버섯의 망간에이즈 퍼옥시다아제(manganese peroxidase)의 유전자(full gene)를 확보하기 위하여 겨울우산버섯을 SSC 배지에서 배양한 후 회수하여 갈고 트리졸(Trizol: invitrogen)을 사용하여 전체 RNA(total RNA)를 분리하였다. cDNA를 합성을 하기 위해 전체 RNA 4㎍을 주형(template)으로 Cap Fishing Full-length cDNA Premix Kit (Seegene)을 사용하여 cDNA를 합성하였다. In order to secure the full gene of manganese peroxidase of winter umbrella mushrooms, winter mushrooms were cultivated in SSC medium, recovered and harvested, and total RNA (total RNA) was obtained using trizol (Trizol: invitrogen). ) Was separated. In order to synthesize cDNA, cDNA was synthesized using Cap Fishing Full-length cDNA Premix Kit (Seegene) as a template with 4 μg of total RNA.

한편, 5'-, 3'-RACE (Rapid Amplification of cDNA Ends)를 수행하기 위하여 각 유전자의 디제너레이트 프라이머(degenerate primer)를 사용하여 확보된 부분 염기서열을 바탕으로 엑손(exon) 부분에서 특이 프라이머(specific primer)를 제작(표 1)하여 UPM 프라이머와 NUP 프라이머와 함께 RACE PCR (1 단계, 25 사이클; 94℃, 30초; 프라이머 어닐링 온도, 30 초; 72℃, 3분)을 수행하였다. On the other hand, in order to perform 5'-, 3'-RACE (Rapid Amplification of cDNA Ends), it is specific in the exon part based on the partial sequencing obtained using degenerate primers of each gene. Specific primers were prepared (Table 1) and RACE PCR (1 step, 25 cycles; 94 ° C., 30 seconds; primer annealing temperature, 30 seconds; 72 ° C., 3 minutes) was performed with UPM and NUP primers. .

디제너레이트 프라이머(degenerate primer): Degenerate primers:

- Forward primerForward primer

· F2 : CACGACGCCATCGSCATCTC F2: CACGACGCCATCGSCATCTC

· F3 : CTYCGCCTSACMTTCCACGAC F3: CTYCGCCTSACMTTCCACGAC

- Reverse primerReverse primer

· R2 : TCGGACTGSAGSCGCAKCTC R2: TCGGACTGSAGSCGCAKCTC

· R6 : BGGRGTKGAGTCGAARGGMGT R6: BGGRGTKGAGTCGAARGGMGT

· R10 : GTCGATGACGGGGAAGAGCGR10: GTCGATGACGGGGAAGAGCG

UPM 프라이머:UPM Primer:

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT+CTAATACGACTCACTATAGGGCCTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT + CTAATACGACTCACTATAGGGC

NUP 프라이머:NUP Primer:

AAGCAGTGGTAACAACGCAGAGTAAGCAGTGGTAACAACGCAGAGT

<실시예 3> 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자 클로닝Example 3 Manganese Peroxidase Gene Cloning

실시예 2에서 분리한 퍼옥시다아제(peroxidase)의 PCR 산물을 pGEM-T Vector system1 kit를 사용하여 벡터에 연결한 후 E. coli에 삽입하였다. 이렇게 확보된 부분 염기서열들을 조합하여 전체길이의 염기서열을 구성하였고 이를 바탕으로 단백질이 만들어지는 코딩 염기서열 (ORF) 부분을 포함해서 다시 프라이머를 제작하 여 PCR (표 2; 3 단계, 29 사이클; 1 단계: 94℃, 2분, 2 단계: 94℃, 30초/ 60℃, 30초/ 72℃, 3분, 1 사이클, 3 단계: 72℃, 10분)을 통해 전체 길이의 cDNA(full length cDNA)를 확보하였다.The PCR product of peroxidase isolated in Example 2 was connected to the vector using pGEM-T Vector system1 kit and then inserted into E. coli . The base sequences of the full length were constructed by combining the partial sequences thus secured and PCR was prepared again by preparing primers including the coding sequence (ORF) portion from which the protein is made, based on the PCR (Table 2; 3 steps, 29 cycles). Step 1: 94 ° C., 2 minutes, step 2: 94 ° C., 30 seconds / 60 ° C., 30 seconds / 72 ° C., 3 minutes, 1 cycle, step 3: 72 ° C., 10 minutes) full length cDNA) was obtained.

<표 1> RACE 프라이머TABLE 1 RACE Primers

CloneClone RACERACE PrimerPrimer Annealing Temp.Annealing Temp. PCR sizePCR size PrimerPrimer sequencesequence pbmnp2pbmnp2 5'5 ' pbmnp1-r1pbmnp1-r1 70.870.8 521521 GAAGGCTATTACACGTACCGAAAGGGAAGGCTATTACACGTACCGAAAGG pbmnp1-r2pbmnp1-r2 74.474.4 422422 CTTGAACTCGAGCTGAGGTGCACCCTTGAACTCGAGCTGAGGTGCACC 3'3 ' pbmnp1-f1pbmnp1-f1 77.877.8 771771 ACGCAAATGGAGGCACCGACGAGACGCAAATGGAGGCACCGACGAG pbmnp1-f2pbmnp1-f2 70.270.2 749749 GATCGTCGCGTTGCAGAAGCCCGATCGTCGCGTTGCAGAAGCCC pbmnp3pbmnp3 5'5 ' pbmnp2-r3pbmnp2-r3 75.475.4 396396 GTGAGGTTGTGGCGCTGGATGAGTGAGGTTGTGGCGCTGGATGA pbmnp2-r2pbmnp2-r2 74.574.5 334334 CGCGTGGAAGTTGGTCTCGATGCGCGTGGAAGTTGGTCTCGATG pbmnp4pbmnp4 5'5 ' pbmnp4-r2pbmnp4-r2 70.770.7 347347 CGTTGTTCGCCGATGATCTCACGTTGTTCGCCGATGATCTCA pbmnp4-r1pbmnp4-r1 71.571.5 329329 CGTGGAAGTTAGTCTCGATGTCGTCCGTGGAAGTTAGTCTCGATGTCGTC pbmnp5pbmnp5 5'5 ' pbmnp7-r2pbmnp7-r2 77.577.5 444444 AGGGCACTGAGCGAGCGCGAAGGGCACTGAGCGAGCGCGA pbmnp7-r3pbmnp7-r3 74.974.9 400400 CGGCCGAGATGTTGTGCTTGGCGGCCGAGATGTTGTGCTTGG 3'3 ' pbmnp7-f4pbmnp7-f4 73.873.8 768768 GAGATCGTCGCGTTGCAGAAGCGAGATCGTCGCGTTGCAGAAGC pbmnp7-f2pbmnp7-f2 74.974.9 737737 TCGCGCTCGCTCAGTGCCCTTCGCGCTCGCTCAGTGCCCT pbmnp7-f3pbmnp7-f3 77.577.5 692692 CCAAGCACAACATCTCGGCCGCCAAGCACAACATCTCGGCCG pbmnp6pbmnp6 5'5 ' pbmnp9-r1pbmnp9-r1 6666 588588 GGCGAGGAGGGCGACAGGCGAGGAGGGCGACA pbmnp9-r3pbmnp9-r3 6969 354354 ATGATCTCATCGACACCAAGGTTCATGATCTCATCGACACCAAGGTTC 3'3 ' pbmnp9-f2pbmnp9-f2 6969 426426 GAACCTTGGTGTCGATGAGATCATGAACCTTGGTGTCGATGAGATCAT pbmnp9-f4pbmnp9-f4 72.572.5 360360 CTTGCTGGTGCGATCGGTGTCCTTGCTGGTGCGATCGGTGTC pbmnp9-f5pbmnp9-f5 73.273.2 309309 GTCTTTGTCGGCCGTCAGGATGGTCTTTGTCGGCCGTCAGGATG

<표 2> Full length cDNA를 위한 PCR 프라이머TABLE 2 PCR primers for full length cDNA

CloneClone PrimerPrimer Full length(ORF)Full length (ORF) pbmnp1pbmnp1 pbmnp1-RT-f1(CGGACTCTACGGACATGG) /pbmnp1-RT-r2(CGAGTGTACAACGTGATCATG)pbmnp1-RT-f1 (CGGACTCTACGGACATGG) / pbmnp1-RT-r2 (CGAGTGTACAACGTGATCATG) 1269(1098) bp1269 (1098) bp pbmnp2pbmnp2 fpbmnp2-f(CAATAGGTCAACGGTCATG) /pbmnp2-RT-r(ACTACAACCTATGTGCTGTTCTG)fpbmnp2-f (CAATAGGTCAACGGTCATG) / pbmnp2-RT-r (ACTACAACCTATGTGCTGTTCTG) 1274(1083) bp1274 (1083) bp pbmnp3pbmnp3 fpbmnp3-f(CACTTCATCTCCTCTTCGG) /pbmnp3-UTR-r(ACTGACAATCTGACTCTCTACGA)fpbmnp3-f (CACTTCATCTCCTCTTCGG) / pbmnp3-UTR-r (ACTGACAATCTGACTCTCTACGA) 1410(1095) bp1410 (1095) bp pbmnp4pbmnp4 fpbmnp4-f-1(CGGAATGCCTCAAACACCCAG) /fpbmnp4-r(TCCAGTCTGAGCAGCACCGC)fpbmnp4-f-1 (CGGAATGCCTCAAACACCCAG) / fpbmnp4-r (TCCAGTCTGAGCAGCACCGC) 1394(1314) bp1394 (1314) bp pbmnp5pbmnp5 fpbmnp5-f(GAGGAACGCATCACCGACAACAG) /fpbmnp5-r(GTTCACCGAGCTGGCTAGCGAG)fpbmnp5-f (GAGGAACGCATCACCGACAACAG) / fpbmnp5-r (GTTCACCGAGCTGGCTAGCGAG) 1471(1098) bp1471 (1098) bp pbmnp6pbmnp6 fpbmnp6-f-1(CAGCTGTTCATCTCCTTCTCGG) /fpbmnp6-r(GCGCCAACAAGTCTAGTCGGTG)fpbmnp6-f-1 (CAGCTGTTCATCTCCTTCTCGG) / fpbmnp6-r (GCGCCAACAAGTCTAGTCGGTG) 1.3kb(1095) bp1.3 kb (1095) bp

SSC 배지에서 배양한 겨울우산버섯(Polyporus brumalis)으로부터 6개의 새로운 망간에이즈 퍼옥시다아제(manganese peroxidase: MnP) 유전자를 분리하였다(도 1). 분리한 유전자를 Pbmnp1 (서열 1), Pbmnp2 (서열 2), Pbmnp3 (서열 3), Pbmnp4 (서열 4), Pbmnp5 (서열 5) 및 Pbmnp6 (서열 6)으로 명명하고 각각 전체 유전자(full gene)의 염기서열을 결정하였다. 도 2는 상기 유전자가 코딩하는 각각의 아미노산 서열(서열 7, 서열 8, 서열 9, 서열 10, 서열 11, 서열 12)을 비교하여 나타낸 것이다. 각각의 MnP 유전자는 각각 51-89% 유사성을 가졌는데 전체 길이는 1,250-1,500bp로 ORF (Open Reading Frame) 부분의 아미노산 서열이 360 aa 정도이고 분자량은 보통의 MnP가 가지는 특성인 38-39 KDa에 포함되었다(표 3). 그러나 UTR (Untranslated Region) 부분의 크기가 매우 달라 발현과 활성에서 다른 특성을 가질 것으로 예상되었다.Six new manganese peroxidase (MnP) genes were isolated from Polyporus brumalis cultured in SSC medium (FIG. 1). The isolated genes were identified as Pbmnp1 (SEQ ID NO: 1), Pbmnp2 (SEQ ID NO: 2), Pbmnp3 (SEQ ID NO: 3), Pbmnp4 (SEQ ID NO: 4), Pbmnp5 (SEQ ID NO: 5) And Pbmnp6 (SEQ ID NO: 6), and the base sequence of the full gene (full gene) was determined, respectively. Figure 2 shows a comparison of each amino acid sequence (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) encoded by the gene. Each MnP gene has 51-89% similarity, and its total length is 1,250-1,500bp. The amino acid sequence of ORF (Open Reading Frame) is about 360 aa and its molecular weight is 38-39 KDa, which is the characteristic of ordinary MnP. (Table 3). However, the size of the UTR (Untranslated Region) region is very different and it is expected to have different characteristics in expression and activity.

<표 3> 겨울우산버섯 MnP 유전자의 특성<Table 3> Characteristics of winter umbrella mushroom MnP

CloneClone Full length(nt)Full length (nt) ORF(aa)ORF (aa) 5'UTR(nt)5'UTR (nt) 3'UTR(nt)3'UTR (nt) Mw.Mw. plpl pbmnp1pbmnp1 1,269bp1,269 bp 365 aa365 aa 17 bp17 bp 154 bp154 bp 38.92 kDa38.92 kDa 4.264.26 pbmnp2pbmnp2 1,274bp1,274 bp 360 aa360 aa 90 bp90 bp 101 bp101 bp 38.48 kDa38.48 kDa 4.844.84 pbmnp3pbmnp3 1,410bp1,410 bp 363 aa363 aa 95 bp95 bp 167 bp167 bp 38.02 kDa38.02 kDa 4.254.25 pbmnp4pbmnp4 1,394bp1,394bp 364 aa364 aa 545 bp545 bp 152 bp152 bp 38.10 kDa38.10 kDa 4.254.25 pbmnp5pbmnp5 1,471bp1,471bp 365 aa365 aa 211 bp211 bp 193 bp193 bp 39.03 kDa39.03 kDa 4.544.54 pbmnp6pbmnp6 1,300bp1,300 bp 364 aa364 aa 77 bp77 bp 128 bp128 bp 37.99 kDa37.99 kDa 4.074.07

<실시예 4> 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자의 발현분석Example 4 Expression Analysis of Manganese Peroxidase Gene

겨울우산버섯의 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자의 발현 분석을 위하여, 겨울우산버섯 균체를 SSC 액체배지 (glucose 1.5 %, ammonium tartrate 0.1 %, pH 4.5, 25℃) 1L에서 5일간 배양하여, 균체를 블렌딩(blending) 한 후 블렌딩한 균사체 500㎖를 새 SSC 배지 500㎖에 계대하였다. 계대 후 5일에 DBP(dibutyl phthalate)를 100uM, 300uM의 농도로 각각 첨가한 다음 9일 후, 15일 후에 균사체를 40㎖씩 회수하였다. 트리졸(Trizol: Invitrogen)을 사용하여 회수한 균사체로부터 total RNA를 분리한 다음 역전사 효소 (Reverse Transcriptase: Promega)를 이용하여 cDNA를 만든 후 표 4와 같은 조건으로 PCR하였다.To analyze the expression of manganese peroxidase gene in winter umbrella, winter mushroom was incubated for 5 days in 1L of SSC liquid medium (glucose 1.5%, ammonium tartrate, pH 4.5, 25 ℃). After blending the cells, 500 ml of the blended mycelium was passaged to 500 ml of fresh SSC medium. After 5 days of passage, DBP (dibutyl phthalate) was added at 100 uM and 300 uM, respectively, and after 9 days, 15 days later, 40 ml of mycelium was recovered. Total RNA was isolated from the mycelium recovered using Trizol: Invitrogen, and cDNA was prepared using Reverse Transcriptase (Promega), and PCR was performed under the conditions shown in Table 4.

<표 4> 발현량 분석에 사용된 프라이머TABLE 4 Primers used for expression analysis

CloneClone ForwardForward ReverseReverse Temp. (℃)Temp. (℃) CyclesCycles pbmnp1pbmnp1 pbmnp1-UTR-f2pbmnp1-UTR-f2 pbmnp1-UTR-r1pbmnp1-UTR-r1 6262 4040 CGTTCCCAATGCATGATC ACCGTTCCCAATGCATGATC AC TACACCGTCCGCCACAACTACACCGTCCGCCACAAC pbmnp2pbmnp2 pbmnp2-UTR-fpbmnp2-UTR-f pbmnp2-UTR-rpbmnp2-UTR-r 5555 4040 TGACCAACGATGACCTTCTGACCAACGATGACCTTC GTGTAAGTCGCCTTTTTCAGGTGTAAGTCGCCTTTTTCAG pbmnp3pbmnp3 pbmnp3-UTR-fpbmnp3-UTR-f pbmnp3-UTR-rpbmnp3-UTR-r 5555 4040 GTGTCAGACCGTGAAGCGGTGTCAGACCGTGAAGCG ACTGACAATCTGACTCTCTACGAACTGACAATCTGACTCTCTACGA pbmnp4pbmnp4 pbmnp4-f1pbmnp4-f1 pbmnp4-UTR-r1pbmnp4-UTR-r1 5555 4040 CTCGCTCGTGACGACTCTCGCTCGCTCGTGACGACTCTCG ACATAATCTCAATGATAGACCAGACATAATCTCAATGATAGACCAG pbmnp5pbmnp5 pbmnp5-UTR-f1pbmnp5-UTR-f1 pbmnp5-UTR-r1pbmnp5-UTR-r1 5555 4040 AACCTATAAGCCGTTCACCAACCTATAAGCCGTTCACC TTCGCTGTAGGGAACATTTCGCTGTAGGGAACAT pbmnp6pbmnp6 pbmnp6-UTR-fpbmnp6-UTR-f pbmnp6-UTR-rpbmnp6-UTR-r 5555 4040 TCCTTCCTAAATATTCACCGTCCTTCCTAAATATTCACCG GTTGGATACGGGTTTACTCAGTTGGATACGGGTTTACTCA

배지 내 효소 활성 변화를 확인하기 위해, 1일부터 20일 동안 배양시킨 각각의 시료에 대해 Whatmann No. 2 여과지를 사용하여 감압 상태에서 여과를 수행하였으며, 이를 조추출액으로 이용하였다. 배지 내 단백질량은 브래드포드 분석(bradford assay) 방법(Bradford, 1976)을 이용하였으며, 리그닌 분해효소로 MnP(manganese-dependent peroxidase)와 라카아제(laccase)의 역가의 활성을 다음과 같은 방법으로 측정하였다. MnP 역가는 ABTS(2,2'-Azino-bis(3- ethylbenzthiazoline -6-sulfonic acid))(0.08 g/ℓ)를 기질로 사용하여 H2O2 (0.1 mM), MnSO4 (0.2 mM) 및 0.2M citrate buffer (pH 4.5) 용액을 이용하여 측정하였으며 조효소액을 제일 나중에 첨가하여 측정을 하였고 ABTS를 기준으로 ∑414= 36,000 M-1㎝-1을 적용하여 계산하였다. To determine the change in enzyme activity in the medium, Whatmann No. Filtration was performed under reduced pressure using two filter papers, which were used as crude extracts. The amount of protein in the medium was determined by Bradford assay (Bradford, 1976), and the activity of MnP (manganese-dependent peroxidase) and laccase (laccase) as lignin degrading enzyme was measured by the following method. It was. MnP titers were determined using H 2 O 2 (0.1 mM), MnSO 4 (0.2 mM) using ABTS (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)) (0.08 g / L) as a substrate. And 0.2M citrate buffer (pH 4.5) solution. The coenzyme solution was added last and measured. Σ 414 = 36,000 M -1 cm -1 was calculated based on ABTS.

겨울우산버섯의 최소배지 배양 시 DBP (Dibutylphthalate) 100μM, 300μM을 각각 더하고 망간에이즈 퍼옥시다아제(manganese peroxidase)의 활성변화 및 발현을 분석한 결과 배양 9일 째 MnP 활성이 발현증가를 보였다. 특히 pbmnp2, pbmnp4 유전자가 강하게 유도되어 환경호르몬 분해에 매우 강하게 발현되는 유전자로 특징지을 수 있었다(도 3a).In the culture of minimally cultured winter umbrellas, DBP (Dibutylphthalate) was added to 100μM and 300μM, respectively, and analysis of the activity and expression of manganese peroxidase showed an increase in MnP activity at 9 days of culture. In particular, pbmnp2 and pbmnp4 genes were strongly induced and could be characterized as genes that are strongly expressed in environmental hormone degradation (FIG. 3A).

내분비장애물질 분해에 대한 겨울우산버섯의 망간에이즈 퍼옥시다아제(manganese peroxidase)의 활성은 DBP(Dibutylphthalate) 무처리(control), 100μM, 300μM을 각각 더하고 측정한 결과 DBP 첨가 시 활성이 무첨가(control) 시보다 더 높음을 알 수 있었고 배양 14일(DBP 첨가 후 9일)에는 100μM 농도에서, 배양 20일(DBP 첨가 후 15일)에는 300μM 농도에서 각각 활성이 높은 것으로 나타났다(도 3b). The activity of manganese peroxidase of winter umbrella on endocrine disruption was measured by adding DBP (Dibutylphthalate) control, 100μM and 300μM, respectively. It was found that the activity was higher at 100 μM concentration at 14 days of culture (9 days after DBP addition) and 300 μM at 20 days of culture (15 days after DBP addition) (FIG. 3B).

<실시예 5> 서던 블럿(Southern blot) Example 5 Southern blot

서던 블럿(Southern blot)에 의해 망간에이즈 퍼옥시다아제(MnP) 유전자인지 여부를 확인하기 위하여 상기 실시예 3에서 분리한 6개의 MnP 유전자를 주 형(template)으로 하여 3-UTR(untranslational region)에 특이적인 프라이머(표 5)를 각각 제작하여 PCR (3 단계, 29 사이클; 1 단계: 94℃, 2분, 2 단계: 사이클, 94℃, 30초/ 프라이머 어닐링 온도, 30초/ 72℃, 30초, 3 단계: 72℃, 10분)로 각 유전자들의 UTR을 증폭하고 TOPO-TA Kit를 사용하여 염기서열을 확인한 후 UTR 부분만 EcoR I 제한효소로 절단하여 약 100ng의 프로브(probe)용 DNA를 확보하였다. 이것을 95℃에서 5 분간 가열하여 단일가닥으로 변성시킨 후 동위원소 32P를 첨가하고 레이블 키트(ladderman labeling Kit: TAKARA)를 이용하여 37℃에서 20분간 반응시켜 32P가 표지된 프로브(probe)를 제작하였다.In order to confirm whether it is a manganese peroxidase (MnP) gene by Southern blot, the six MnP genes isolated in Example 3 were used as a template, which was specific to 3-UTR (untranslational region). Specific primers (Table 5) were prepared by PCR (3 steps, 29 cycles; 1 step: 94 ° C., 2 minutes, 2 steps: cycle, 94 ° C., 30 seconds / primer annealing temperature, 30 seconds / 72 ° C., 30 seconds) , Step 3: 72 ℃, 10 minutes) to amplify the UTR of each gene and confirm the nucleotide sequence using the TOPO-TA Kit and then cut only the UTR portion with Eco RI restriction enzyme to the DNA for about 100ng of probe (probe) Secured. A is 32 P labeled by 20 min at 37 ℃ using: (TAKARA ladderman labeling Kit) probe (probe) was heated at 95 ℃ 5 minutes with the addition of the isotope 32 P was modified with single-strand and label kit Produced.

<표 5> 프로브(Probe) 제작에 사용된 프라이머<Table 5> Primers used to make probes

CloneClone PrimerPrimer SizeSize Annealing temp.Annealing temp. pbmnp1pbmnp1 pbmnp1-UTR-f2(CGTTCCCAATGCATGATCAC) /pbmnp1-UTR-r1(TACACCGTCCGCCACAAC)pbmnp1-UTR-f2 (CGTTCCCAATGCATGATCAC) / pbmnp1-UTR-r1 (TACACCGTCCGCCACAAC) 152bp152 bp 62℃ 62 ℃ pbmnp2pbmnp2 pbmnp2-UTR-f(TGACCAACGATGACCTTC) /pbmnp2-UTR-r(GTGTAAGTCGCCTTTTTCAG)pbmnp2-UTR-f (TGACCAACGATGACCTTC) / pbmnp2-UTR-r (GTGTAAGTCGCCTTTTTCAG) 101bp101 bp 55℃ 55 ℃ pbmnp3pbmnp3 pbmnp3-UTR-f(GTGTCAGACCGTGAAGCG) /pbmnp3-UTR-r(ACTGACAATCTGACTCTCTACGA)pbmnp3-UTR-f (GTGTCAGACCGTGAAGCG) / pbmnp3-UTR-r (ACTGACAATCTGACTCTCTACGA) 138bp138 bp 55℃ 55 ℃ pbmnp4pbmnp4 pbmnp4-UTR-f1(TAAAGTGTCGCGGTGCTG) /pbmnp4-UTR-r1(ACATAATCTCAATGATAGACCAG)pbmnp4-UTR-f1 (TAAAGTGTCGCGGTGCTG) / pbmnp4-UTR-r1 (ACATAATCTCAATGATAGACCAG) 401bp401 bp 55℃ 55 ℃ pbmnp5pbmnp5 pbmnp5-UTR-f1(AACCTATAAGCCGTTCACC) /pbmnp5-UTR-r1(TTCAAAGCTGTAGGGAACAT)pbmnp5-UTR-f1 (AACCTATAAGCCGTTCACC) / pbmnp5-UTR-r1 (TTCAAAGCTGTAGGGAACAT) 144bp144bp 55℃55 ℃ pbmnp6pbmnp6 pbmnp6-UTR-f(TCCTTCCTAAATATTCACCG) /pbmnp6-UTR-r(GTTGGATACGGGTTTACTCA)pbmnp6-UTR-f (TCCTTCCTAAATATTCACCG) / pbmnp6-UTR-r (GTTGGATACGGGTTTACTCA) 100bp100bp 55℃55 ℃

겨울우산버섯의 게놈 DNA(genomic DNA)는 회수된 균사체로부터 세틸트리메틸암모니움브로마이드(cetyltrimethylammonium bromide: CTAB)를 이용한 방법에 의해 분리하였다. 분리한 게놈 DNA를 4개의 마이크로 튜브에 25㎍씩 분취하여 4 종의 제한효소 (Xho I, BamH I, EcoR V, Pst I) 50 unit 으로 24시간 동안 37℃에 반응시 켜 절단한 후, 0.8 % 아가로스 겔 상에서 3시간 도안 80 V로 전기영동 하여 게놈 DNA 절편들을 전개시켰다. 전기영동 겔을 250 mM HCl에서 20분간 침지하여 디퓨리네이션(defurination) 시키고 변성(denature) 용액(0.5 M NaOH2, 1.5 M NaCl)에 15분간 두 번 침지시킨 후, 중화(neutralization) 용액 (0.5 M Tris-HCl(pH 7.5), 0.1 M NaCl)에 두 번 침지시킨 다음 증류수로 두 번 씻은 뒤 20×SSC 버퍼 (3 M NaCl, 0.3 M Sodium citrate, pH 7.0)로 제타-프로브 GT 멤브레인(Zeta-probe GT membrane; Bio-Rad)에 블럿(blotting) 하였다. Genomic DNA of the winter umbrella was isolated from the recovered mycelium by the method using cetyltrimethylammonium bromide (CTAB). 25 μg of the genomic DNA was separated into four microtubes and digested with 50 units of four restriction enzymes ( Xho I, Bam H I, Eco R V, Pst I) at 37 ° C. for 24 hours, and then 0.8. Genomic DNA fragments were developed by electrophoresis at 80 V for 3 hours on a% agarose gel. The electrophoretic gel was soaked in 250 mM HCl for 20 minutes for defurination and then immersed twice in denatured solution (0.5 M NaOH 2, 1.5 M NaCl) for 15 minutes and then neutralized solution (0.5 M). Dipping twice in Tris-HCl (pH 7.5), 0.1 M NaCl), rinsing twice with distilled water and then zeta-probe GT membrane (Zeta-) with 20 × SSC buffer (3 M NaCl, 0.3 M Sodium citrate, pH 7.0) The probe GT membrane (Bio-Rad) was blotted.

블럿(Blot)은 3시간 동안 40℃에서 하이브리다이제이션(hybridization) 용액으로 프리-하이브리다이제이션(pre-hybridization) 하고, 각 유전자 cDNA의 3-UTR(untranslational region) 부분에 특이적인 32P-표지한 프로브(32P-labeled probe)와 하이브리다이제이션 하였다. 하이브리다이제이션은 42℃하에 pH 7.2의 0.5 M 소듐 포스페이트(sodium phosphate), 7% SDS, 1 mM EDTA 조건으로 수행하였다. 하이브리드(Hybrid)된 멤브레인은 워싱 버퍼 Ⅰ(washing buffer Ⅰ: 2X SSC, 0.2% SDS)로 두 번 실온에서 워싱한 후 워싱 버퍼 Ⅱ(washing buffer Ⅱ: 0.2X SSC, 0.2% SDS)로 62℃에서 반복해서 워싱하였다. 멤브레인을 비닐 백에 넣어서 물기가 흐르지 않도록 봉한 다음 암실에서 X-ray 필름과 함께 카세트에 넣어서 -70℃에서 하루 동안 노출(expose)시킨 후 X-ray 필름을 현상(develope) 용액과 고정(fixer) 용액에서 현상시킨 후, 물로 세척하여 말려서 밴드(band)를 확인하였다. Blots were pre-hybridized with a hybridization solution at 40 ° C. for 3 hours and 32 P-labels specific for the 3-UTR (untranslational region) portion of each gene cDNA. One probe ( 32 P-labeled probe) was hybridized with. Hybridization was performed at 42 ° C. with 0.5 M sodium phosphate, 7% SDS, 1 mM EDTA at pH 7.2. Hybrid membranes were washed twice at room temperature with washing buffer I (2X SSC, 0.2% SDS) and then at 62 ° C. with washing buffer II (0.2X SSC, 0.2% SDS). Washed repeatedly. The membranes are sealed in a plastic bag to prevent moisture, and then placed in a cassette with an X-ray film in the dark, exposed at -70 ° C. for one day, and the X-ray film fixed with a developer solution. After developing in solution, the solution was washed with water and dried to confirm a band.

겨울우산버섯의 크로모좀(chromosomal) DNA로부터 유래된 유전자임을 확인하 기 위하여 4가지 제한 효소에 의해 각각의 카피(copy) 수가 보이는지 확인하였고 각각의 유전자들이 다른 밴드의 양상을 띠는 것을 보아 각각의 유전자들이 확실히 다른 특성을 가지고 있다고 결론지을 수 있었다(도 4). In order to confirm that the gene is derived from the chromosomal DNA of the Umbrella, the number of copies was shown by four restriction enzymes. It could be concluded that the genes certainly have different characteristics (FIG. 4).

도 1은 겨울우산버섯의 MnP 유전자를 클로닝 한 후 전기영동 하여 확인한 것이다.1 is confirmed by electrophoresis after cloning the MnP gene of the winter umbrella.

도 2는 겨울우산버섯의 MnP 유전자의 alignment를 나타낸 것이다.Figure 2 shows the alignment of the MnP gene of the winter umbrella.

도 3a는 겨울우산버섯의 MnP 발현 특성을 나타낸 것이다.Figure 3a shows the MnP expression characteristics of the winter umbrella.

도 3b는 겨울우산버섯의 MnP 활성을 측정하여 나타낸 것이다.Figure 3b shows the measurement of the MnP activity of the winter umbrella.

도 4는 Southern blot 분석에 의한 겨울우산버섯의 MnP를 확인한 것이다.Figure 4 confirms the MnP of the winter umbrella by Southern blot analysis.

<110> Korea Forest Research Institute <120> Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1316 <212> DNA <213> Polyporus brumalis <400> 1 caccggactc tacggacatg gccttccaag ctctcctctc catagtcgcc gttgttgctg 60 cccttcaagg cgtcgacgct gcgctcaccc gccgcgtcgc ttgcccggac ggcaagaaca 120 ccgcgacgaa cgcggcatgc tgcgccctct tccctatccg ggacgacatc gtcgagaacc 180 tcttccacaa ccagtgcgct gaggaggccc acgagtccct ccgcttgact ttccacgacg 240 ccgtcgcctt ctcgcccgcg gcgcaggccc ggggcgagtt tgtcggcgga ggcgctgacg 300 gctccatcgt cctcttctcc gacatcgaga cgaacttcca tgcgaacgac ggcaccgacg 360 acatcgtcgc gttgcagaag ccgttcatcg ccaagcacaa catctctgcc gctgacttcg 420 tgcaatttgc gggtgccgtc gggctcgctc agtgccccgg tgcacctcag ctcgagttcc 480 tgctcggtcg caaggatgcc actcagcctg ctaatgatgg tcttgttcca gagcccttcg 540 atagcgttga cagcatcctc gctcgcctcc tcgacgtcgg cttccaggac ttcgaggtcg 600 tctggctgct ctccgcccat acggtcgcag ctgccgacag ggtggacccc accatcccgc 660 gcacgccctt cgattctacc ccggagatct tcgacaccca gttcttcatc gagactcagc 720 tccgcgggca gacattcccc ggcgtgggcg gcgagcaggg cgaggtgacg tccccgctcc 780 gcggcgagat gcggctgcaa tcagaccacc tcctcgcacg cgactcgcgc acctcgtgcg 840 agtggcagtc tttcaccaac gaccaggaga agttcgccga gacgttcccg gacgtcatgg 900 gccgccttgc gctcctcggc gtggaccaga gccagctcat cgactgctcg gaagtcatcc 960 ccatcgcgcc gcctctcccc gccagctccc gcccccactt ccccgctggc aagactgctg 1020 ctgatgtcga gcaggcttgc gccgagaccc ccttcccgtc cttccccacc gaccccggcc 1080 cggccacctc tgttgcgccc gttcccaatg catgatcacg ttgtacactc gtacacgacc 1140 cggagtattc gtgttggccc attgtatgct tttcgacttg tggtgtacta catacttagt 1200 ggtatcagga ccgtcgcagc tgcgcccggc gctggttgtg gcggacggtg tacattacct 1260 cgtgtgtgaa acttgtatca ctgtcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1316 <210> 2 <211> 1226 <212> DNA <213> Polyporus brumalis <400> 2 caataggtca acggtcatgg ctttcaaggc tctgctctcc ttcgtctcca tcgtcgccgc 60 catccagggt gccaccgctg cgctcacaag gcgcgtcgct tgccccgacg gcaagaacac 120 tgcgaccaat gcagcgtgct gcgccctctt ccccattcgg gacgacatcg tcaagaacct 180 cttccacaac cagtgcgcag aggaggccca cgagtccctc cgcctcacct tccacgacgc 240 tgtcgcgttc tcgcccgctg cccaagcccg tggccagttc gccggtggag gcgccgacgg 300 ctccatcgtt ctcttctccg aaatcgagac ggccttccac gcaaatggag gcaccgacga 360 gatcgtcgcg ttgcagaagc ccttcatcgc caagcataac atctccgccg ctgacttcgt 420 tcaattcgcg ggcgccgtcg ggctcgctca gtgccccggt gcacctcagc tcgagttcaa 480 gctcggtcgc aaggacgcaa cccgggccgc acctgacggt ctcgtccccg aacctttcga 540 ctcggtcgac aagatccttg cccgcttgct cgacgtcggt ttccaggact tcgaggtcgt 600 atggctgctc tccgcacaca ctgtcgccgc cgccgaccta gtcgacccga cgatcccgcg 660 tacgccattc gactcaactc cggaaatctt cgacacccag tttttcatcg agacccagct 720 ccgcggacag accttccccg gtaagggcgg catccagggc gaggtgacgt ccccgctccg 780 cggcgagatg cgcctgcagt ccgaccatct gctcgcgcgt gactcgcgca catcgtgcga 840 gtggcagtcg ttcaccaacg accaggagaa gtttgcggag acgttcccgg acgtcatggg 900 ccgccttgcg ctcctcggtg tcgacgagag caagctcatt gattgctcgg aggtcattcc 960 gctcgccccg cccctcccgg ccaacatccg cccgcatttc cctgctggaa agacccatgc 1020 cgacatcgag caagcatgtg ctgagacgcc gttcccctcg ggtccggcga ctgtcgtcgc 1080 tcctgttccc aacctttaga ttggtccagg ctctgaaggt catcgttggt cacaggttca 1140 tagacagtgt catggtgctg cagtatatga gtggtcactc gcgaatacta tctgacttat 1200 gatcatcaaa aaaaaaaaaa aaaaaa 1226 <210> 3 <211> 1905 <212> DNA <213> Polyporus brumalis <400> 3 ggcacgaggg ttagacagac atgctgtctg cccgcactgt cttgcaattc cgtatacaac 60 accaacgttc cagggagtgt actctcacca cgaacctcac ctgcccggtg ccggtcctgc 120 ggagcaccca ctgccgtgac tcgggcatcc gctccactgt cgcatcggta ccgtggtcgt 180 actcgcaggc atcggcacca ttcgcacact tgtttggtac cagctcctac gctgcccaca 240 gccgaggtgc cgacaccgtc ctaaatatct ggttctctca ggagcgcacc cgaaccctgg 300 tagaggtgct atgcaagcag cgagagacat gacccgccgg gtgcccgacg caccgtttgg 360 gttaggcagc gacgtgccat tggctgcgtc catattgtac gccgacacgc cgacgtcggc 420 atgccctgta cacccatggc atcgggttat tgcgttcacg ctgtcagaat gggtgcagcg 480 tgattggctg gcctccttgg caagggttta tgtacctttc gcaatttcga ggacatagca 540 gcaccttttg caggatggcg aagagtataa aagagctgtg gcgactgcca ccaaacttca 600 ggacatccac ctttcctcag tcacttcatc tcctcttcgg acaatggcct tcaaaacact 660 cgcgtctttc gtctcggttc ttgcggccct ccaggtcgcc aacggtgccc tcgtacgccg 720 ggtcacttgc gctgacggca gcgtcacgtc caacgcggca tgctgcgcgc tcttcccggt 780 gatccaggat ctccagacaa acctcttcga cggcggcgag tgtggtgagg aggttcacga 840 atcccttcgt ctgaccttcc acgacgccat cggtatctct cccgccatcg cctcccgcgg 900 ccaattcggt ggtggaggtg ccgacggttc gatcgccctc ttcgaagaca tcgagaccaa 960 cttccacgcg aacaacggtg tcgacgagat catcgacgaa cagcgccccc tcatccagcg 1020 ccacaacctc accacggctg acttcatcca gcttgctggt gccatcggtg tcagcaactg 1080 tcctggtgcc cctcgtcttg cagtgttcgt tggtcgcccc gacgccacac agcccgcgcc 1140 cgacctgacc gtcccggagc ccttcgacac cgtcgacagc atcctgcaac gcttcgatga 1200 tgccggtggc ttcactccag cggaggtcgt ggcgctcctc ggctcccaca ccatcgccgc 1260 ggccgaccac gtcgacccgt ccatcccggg aactcccttc gactcgactc ctgagctctt 1320 cgacacccag ttctttatcg agactcagct ccgtggcacg ctcttccccg gcaccggcgg 1380 caaccagggt gaggtcgagt cgcccctccg cggcgagctc cgtctccagt ccgactccga 1440 gctcgcccgt gactctcgca ctgcttgcga gtggcagtcc ttcgtcaaca accaggctaa 1500 gctccagtcc gcgttcgctg ctgccttccg caagatgacg atcctcgggc acgacgaaag 1560 ctcgctgatc gactgttctg acgtcgtccc gactcccccg gccccggcct ccagcgcgca 1620 ccttcccgcc ggcctcaccc acaacgacgt cgagcaggct tgcgccagca cccccttccc 1680 cacgctcccc accgaccccg gtccggccac cactgtcgct cctgtccctc cctcttagag 1740 tgtgtcagac cgtgaagcgg tgctcaggct ggagaacgtc tttcattgtc cgggcgttgc 1800 tggaatgtcg ccacgtatct ttgaccatcg ggtgtatttc tatgtatcat gggctatcgt 1860 agagagtcag attgtcagta aaaaaaaaaa aaaaaaaaaa aaaaa 1905 <210> 4 <211> 2011 <212> DNA <213> Polyporus brumalis <400> 4 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 a 2011 <210> 5 <211> 1502 <212> DNA <213> Polyporus brumalis <400> 5 acgcggggtc agggaggaac gcatcaccga caacagcaaa tggtcgtgtt gtcattggtc 60 acccttcgag tatccgacca tggtgtaacg actcagcgag atacaggacg gtcacgtccg 120 gctcatatgc agagtccgcc agggtataag ggcctcctcc ttcgtccctc cactcctcag 180 cgaactcggc tttcgttacc agaagcacga catgggtttc aaggctctcc tctctattct 240 cgccgtcgtc gcaaggctcg aggtcgccag cgctgcgctt acgcgccgtg ttgcgtgccc 300 cgacgggaag aacaccgcga cgaacgcggc gtgctgcgct ctgttcccga tccgggacga 360 catcgtcgag aacctcttcc acaaccagtg cgccgaggag gcccacgaat ccctccgtct 420 gaccttccac gacgctgtcg cgttctcgcc cgcagctgag gctcgcggcc agttcgccgg 480 tggaggcgcc gacggctcca tcgttctctt ctccgagatc gagacggcat tccacgcaaa 540 cggaggcacc gacgagatcg tcgcgctgca gaagcccttc atcgccaagc acaacatctc 600 ggccgctgac ttcattcaat tcgcgggcgc cgtcgcgctc gctcagtgcc ctggtgcacc 660 tcagctcgag ttcatgctcg gccgcaagga tgctacgcgg gccgcccctg atggtctcgt 720 ccccgagccc ttcgacacaa tcgatgacat cctcgcccgc ttgctcgacg tcgggttcca 780 ggacttcgaa gtcgtgtggc tgctctcagc acacaccgtc gctgccgccg acctggtcga 840 cccgaccatc ccgcgcacgc ccttcgactc gacgccggag atctttgaca cccagttctt 900 catcgagacc cagctccgtg gtctgaagtt cgccggccag ggcggcatcc acggcgaggt 960 caactctccg ctcaccggcg agatgcgcct ccagtcggac cacctcctcg ctcgcgactc 1020 gcgcacggcc tgcgagtggc agtcattcac gaacgaccag gagaagttcg cggagacgtt 1080 cccggatgtg atgggccgcc ttgcgctcct cggtgttgat cagagcacgc tcatcgactg 1140 ctctgaggtc atcccgcttg ccccgcccct gcccgccagc tctcgcccgc acttccccgc 1200 tggcaagacc cacgccgaca tcgaacaggc ttgcgccgac accccgttcc cgaccttccc 1260 cactgaccct ggtccggcca ccaaggtcgc ccccgttccc aacctataag ccgttcaccg 1320 agctggctag cgagcgtcat gaggtccatt tgcgggctac aagagcacac agcaggggtg 1380 ttgctggttg taggtgccgc tggttataga tgctatcgga gcccatgttc cctacagctt 1440 tgaagttatg ttgaataacg aatatcaaac gtttgaattt caaaaaaaaa aaaaaaaaaa 1500 aa 1502 <210> 6 <211> 2245 <212> DNA <213> Polyporus brumalis <400> 6 aagcagtggt aacaacgcag agtacgcggg ataggctcaa gttgtattcc acggccgtgg 60 gccacaaggt tcccgcagca ggacttttac tgctttgtga cgcggccgtg ggagcggtac 120 atctcgggaa gctcaagcta aaatatggcc gacacggcag gatcttaagg ctttgggtcc 180 tgcttcgatc gtgccatctc cgggcctggg acgcatgccc gaggccggag ctcgagctcg 240 aaccacatcg ccgtttctgg tgaccccacc cagagcccct gcgatttgga cctgccaagg 300 gacctacaat cgcacgaacg tcaacttggc gtgtcgtttt cctttttcct tgtgccatag 360 cagtgcagcg gtctgcgcgg tgtacgcctg catgtccgaa gatagagctt cagactcaag 420 ctgtgcactc tcaccaagaa cctctcgctc gccgttgccg aagaccagca tcctggtata 480 tccccatact accgctccac ccgttcgttc cttgacatgg cagccccacc gtggcaatgg 540 agctgtggtt gaggtcgcag gaactgggtg cggcccgcta ccgccttgtt ccatctgcaa 600 tgccgcccgc agccgaggac tcgacgcgcc cgcccatgcc tccgtttctc atgaacgcgc 660 ccttcgcctg gtggacgtgt cgcgtaaata agtccggaac atgtcgctct gggtacccgc 720 tctacggtct tggtcgcccg ccgtgcttca ttggcccgcc gaccatcacg gaccgacaca 780 ccgacgctat ggactaactt ctatatccca tgcgtcggcc agtccattca cgcatgtgaa 840 tgcgcgctcg gcgattggcc gacctcttgg ttttggggtc gacctaccaa gggtataagc 900 tggacgacac tgcaggtaac caattttgca tgcggacgag gacgtataaa gaggctggta 960 cgacggccag caagattcag gacacctacc tcctcctcag ctgttcatct ccttctcgga 1020 caatggcatt cgcaatcctc gcttccttcg tctctgccct cgcggctctt caggtcgcca 1080 acggtgccct tactcgccgg gtgacctgcg ccgacggcag cgtgacgtcc aacgcggcat 1140 gctgcgcgct cttcccggtc attcaggacc tccaggctaa cctcttcgac aacggtggat 1200 gtggcgagga cgtccacgag tcccttcgct tgaccttcca cgacgccatt ggtatatccc 1260 ctgccatcgc cgctcgcggg caattcggtg gcggaggcgc cgacggctcg atcgccctgt 1320 tcgaggacat cgagaccaac ttccacgcga accttggtgt cgatgagatc atcgacgagc 1380 agcgtcccct cctccagcgc tccaacatca gcgcggctga cttcattcag cttgctggtg 1440 cgatcggtgt cgccaactgc cccggtgctc ctcgcctcgc cgtctttgtc ggccgtcagg 1500 atgctactca gcccgcaccc gacttgacag tccctgagcc tttcgacacc gtcgacagca 1560 tcctgcagcg cttcgaggat gccggtggct tcacccccgc tgaagttgtc gccctcctcg 1620 cctctcacac gatcgctgca gccgaccacg tggacccgag catccccgga acgcctttcg 1680 actccacgcc tgagttgttc gacacccagt tcttcatcga gacccagctc cgcggcaccc 1740 tcttccccgg aaccccaggt aaccagggcg aggtcgagtc accagtcccc ggcgagatcc 1800 gtctccagtc tgactctgag ctcgctcgcg actctcgcac tgcttgcgag tggcagtcct 1860 ttgtcaacaa ccaggctaag ctccagtccg cgttcgccgc tgcgttccgc aagatgacga 1920 tcctcggcca cgacgagagc gagctcgtcg actgctccga cgtcgtccag cagcccccgc 1980 ctccggcttc cgctgcgcac ttccccgccg gtctcagcaa cgccgacatc gagcaggcgt 2040 gcgccagcac tcccttcccc actctcccca ccgaccccgg cccggtcacc tcggtcgcac 2100 ctgtccctcc ttcctaaata ttcaccgact agacttgttg gcgctcttga agggttctga 2160 agttctccct gacgtctcgt gtatatatga gtaaacccgt atccaacctg cttctctnta 2220 gcaaaaaaaa aaaaaaaaaa aaaaa 2245 <210> 7 <211> 365 <212> PRT <213> Polyporus brumalis <400> 7 Met Ala Phe Gln Ala Leu Leu Ser Ile Val Ala Val Val Ala Ala Leu 1 5 10 15 Gln Gly Val Asp Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly 20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg 35 40 45 Asp Asp Ile Val Glu Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala 50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro 65 70 75 80 Ala Ala Gln Ala Arg Gly Glu Phe Val Gly Gly Gly Ala Asp Gly Ser 85 90 95 Ile Val Leu Phe Ser Asp Ile Glu Thr Asn Phe His Ala Asn Asp Gly 100 105 110 Thr Asp Asp Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn 115 120 125 Ile Ser Ala Ala Asp Phe Val Gln Phe Ala Gly Ala Val Gly Leu Ala 130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Leu Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Gln Pro Ala Asn Asp Gly Leu Val Pro Glu Pro Phe Asp Ser 165 170 175 Val Asp Ser Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe 180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Arg 195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile 210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Gln Thr Phe 225 230 235 240 Pro Gly Val Gly Gly Glu Gln Gly Glu Val Thr Ser Pro Leu Arg Gly 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr 260 265 270 Ser Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu 275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Gln 290 295 300 Ser Gln Leu Ile Asp Cys Ser Glu Val Ile Pro Ile Ala Pro Pro Leu 305 310 315 320 Pro Ala Ser Ser Arg Pro His Phe Pro Ala Gly Lys Thr Ala Ala Asp 325 330 335 Val Glu Gln Ala Cys Ala Glu Thr Pro Phe Pro Ser Phe Pro Thr Asp 340 345 350 Pro Gly Pro Ala Thr Ser Val Ala Pro Val Pro Asn Ala 355 360 365 <210> 8 <211> 360 <212> PRT <213> Polyporus brumalis <400> 8 Met Ala Phe Lys Ala Leu Leu Ser Phe Val Ser Ile Val Ala Ala Ile 1 5 10 15 Gln Gly Ala Thr Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly 20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg 35 40 45 Asp Asp Ile Val Lys Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala 50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro 65 70 75 80 Ala Ala Gln Ala Arg Gly Gln Phe Ala Gly Gly Gly Ala Asp Gly Ser 85 90 95 Ile Val Leu Phe Ser Glu Ile Glu Thr Ala Phe His Ala Asn Gly Gly 100 105 110 Thr Asp Glu Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn 115 120 125 Ile Ser Ala Ala Asp Phe Val Gln Phe Ala Gly Ala Val Gly Leu Ala 130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Lys Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Arg Ala Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Ser 165 170 175 Val Asp Lys Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe 180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Leu 195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile 210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Gln Thr Phe 225 230 235 240 Pro Gly Lys Gly Gly Ile Gln Gly Glu Val Thr Ser Pro Leu Arg Gly 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr 260 265 270 Ser Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu 275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Glu 290 295 300 Ser Lys Leu Ile Asp Cys Ser Glu Val Ile Pro Leu Ala Pro Pro Leu 305 310 315 320 Pro Ala Asn Ile Arg Pro His Phe Pro Ala Gly Lys Thr His Ala Asp 325 330 335 Ile Glu Gln Ala Cys Ala Glu Thr Pro Phe Pro Ser Gly Pro Ala Thr 340 345 350 Val Val Ala Pro Val Pro Asn Leu 355 360 <210> 9 <211> 364 <212> PRT <213> Polyporus brumalis <400> 9 Met Ala Phe Lys Thr Leu Ala Ser Phe Val Ser Val Leu Ala Ala Leu 1 5 10 15 Gln Val Ala Asn Gly Ala Leu Val Arg Arg Val Thr Cys Ala Asp Gly 20 25 30 Ser Val Thr Ser Asn Ala Ala Cys Cys Ala Leu Phe Pro Val Ile Gln 35 40 45 Asp Leu Gln Thr Asn Leu Phe Asp Gly Gly Glu Cys Gly Glu Glu Val 50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Ile Gly Ile Ser Pro 65 70 75 80 Ala Ile Ala Ser Arg Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser 85 90 95 Ile Ala Leu Phe Glu Asp Ile Glu Thr Asn Phe His Ala Asn Asn Gly 100 105 110 Val Asp Glu Ile Ile Asp Glu Gln Arg Pro Leu Ile Gln Arg His Asn 115 120 125 Leu Thr Thr Ala Asp Phe Ile Gln Leu Ala Gly Ala Ile Gly Val Ser 130 135 140 Asn Cys Pro Gly Ala Pro Arg Leu Ala Val Phe Val Gly Arg Pro Asp 145 150 155 160 Ala Thr Gln Pro Ala Pro Asp Leu Thr Val Pro Glu Pro Phe Asp Thr 165 170 175 Val Asp Ser Ile Leu Gln Arg Phe Asp Asp Ala Gly Gly Phe Thr Pro 180 185 190 Ala Glu Val Val Ala Leu Leu Gly Ser His Thr Ile Ala Ala Ala Asp 195 200 205 His Val Asp Pro Ser Ile Pro Gly Thr Pro Phe Asp Ser Thr Pro Glu 210 215 220 Leu Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Thr Leu 225 230 235 240 Phe Pro Gly Thr Gly Gly Asn Gln Gly Glu Val Glu Ser Pro Leu Arg 245 250 255 Gly Glu Leu Arg Leu Gln Ser Asp Ser Glu Leu Ala Arg Asp Ser Arg 260 265 270 Thr Ala Cys Glu Trp Gln Ser Phe Val Asn Asn Gln Ala Lys Leu Gln 275 280 285 Ser Ala Phe Ala Ala Ala Phe Arg Lys Met Thr Ile Leu Gly His Asp 290 295 300 Glu Ser Ser Leu Ile Asp Cys Ser Asp Val Val Pro Thr Pro Pro Ala 305 310 315 320 Pro Ala Ser Ser Ala His Leu Pro Ala Gly Leu Thr His Asn Asp Val 325 330 335 Glu Gln Ala Cys Ala Ser Thr Pro Phe Pro Thr Leu Pro Thr Asp Pro 340 345 350 Gly Pro Ala Thr Thr Val Ala Pro Val Pro Pro Ser 355 360 <210> 10 <211> 437 <212> PRT <213> Polyporus brumalis <400> 10 Met Pro Gln Thr Pro Arg Glu Ser Gly Ala Cys Val His Ala Val Arg 1 5 10 15 Met Gly Ala Ala Arg Leu Ala Gly Leu Leu Glu His Asp Leu Arg Leu 20 25 30 Ala Arg Ser Arg His Ser Ile Thr Val Lys Ala Leu Ala Asn Glu Tyr 35 40 45 Lys Gly Ala Gly Ala Thr Thr Thr Lys Pro Gln Asp Ile Tyr Leu Ser 50 55 60 Ser Val Ala Pro Ser Pro Leu Arg Thr Met Ala Phe Lys Thr Leu Ala 65 70 75 80 Ser Phe Val Ser Val Leu Ala Ala Phe Gln Ala Ala Asn Gly Ala Leu 85 90 95 Val Arg Arg Val Thr Cys Ala Asp Gly Ser Val Thr Ala Asn Ala Ala 100 105 110 Cys Cys Ala Leu Phe Pro Val Ile Gln Asp Leu Gln Thr Asn Leu Phe 115 120 125 Asp Gly Gly Glu Cys Gly Glu Glu Val His Glu Ser Leu Arg Leu Thr 130 135 140 Phe His Asp Ala Ile Gly Ile Ser Pro Ala Ile Ala Ser Arg Gly Gln 145 150 155 160 Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ala Ile Phe Asp Asp Ile 165 170 175 Glu Thr Asn Phe His Ala Asn Asn Gly Val Asp Glu Ile Ile Gly Glu 180 185 190 Gln Arg Pro Leu Ile Gln Arg His Asn Ile Thr Thr Ala Asp Phe Ile 195 200 205 Gln Leu Ala Gly Ala Ile Gly Val Ser Asn Cys Pro Gly Ala Pro Arg 210 215 220 Leu Ala Val Phe Val Gly Arg Pro Asp Ala Thr Gln Pro Ala Pro Asp 225 230 235 240 Leu Thr Val Pro Glu Pro Phe Asp Thr Val Asp Ser Ile Leu Gln Arg 245 250 255 Phe Asp Asp Ala Gly Gly Phe Thr Pro Glu Glu Val Val Ala Leu Leu 260 265 270 Gly Ser His Thr Ile Ala Ala Ala Asp His Val Asp Pro Ser Ile Pro 275 280 285 Gly Thr Pro Phe Asp Ser Thr Pro Glu Leu Phe Asp Thr Gln Phe Phe 290 295 300 Ile Glu Thr Gln Leu Arg Gly Thr Leu Phe Pro Gly Thr Gly Gly Asn 305 310 315 320 Gln Gly Glu Val Glu Ser Pro Leu Arg Gly Glu Leu Arg Leu Gln Ser 325 330 335 Asp Ser Glu Leu Ala Arg Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser 340 345 350 Phe Val Asn Asn Gln Ala Lys Leu Gln Ser Ala Phe Ala Ala Ala Phe 355 360 365 Arg Lys Met Thr Ile Leu Gly His Asp Glu Gly Ser Leu Val Asp Cys 370 375 380 Ser Asp Val Val Pro Thr Pro Pro Ala Pro Ala Ser Gln Ala His Leu 385 390 395 400 Pro Ala Gly Leu Thr His Asn Asp Val Glu Gln Ala Cys Ala Ser Thr 405 410 415 Pro Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Ala Thr Ser Val Ala 420 425 430 Pro Val Pro Pro Ser 435 <210> 11 <211> 365 <212> PRT <213> Polyporus brumalis <400> 11 Met Gly Phe Lys Ala Leu Leu Ser Ile Leu Ala Val Val Ala Arg Leu 1 5 10 15 Glu Val Ala Ser Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly 20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg 35 40 45 Asp Asp Ile Val Glu Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala 50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro 65 70 75 80 Ala Ala Glu Ala Arg Gly Gln Phe Ala Gly Gly Gly Ala Asp Gly Ser 85 90 95 Ile Val Leu Phe Ser Glu Ile Glu Thr Ala Phe His Ala Asn Gly Gly 100 105 110 Thr Asp Glu Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn 115 120 125 Ile Ser Ala Ala Asp Phe Ile Gln Phe Ala Gly Ala Val Ala Leu Ala 130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Met Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Arg Ala Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Thr 165 170 175 Ile Asp Asp Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe 180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Leu 195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile 210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Leu Lys Phe 225 230 235 240 Ala Gly Gln Gly Gly Ile His Gly Glu Val Asn Ser Pro Leu Thr Gly 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr 260 265 270 Ala Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu 275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Gln 290 295 300 Ser Thr Leu Ile Asp Cys Ser Glu Val Ile Pro Leu Ala Pro Pro Leu 305 310 315 320 Pro Ala Ser Ser Arg Pro His Phe Pro Ala Gly Lys Thr His Ala Asp 325 330 335 Ile Glu Gln Ala Cys Ala Asp Thr Pro Phe Pro Thr Phe Pro Thr Asp 340 345 350 Pro Gly Pro Ala Thr Lys Val Ala Pro Val Pro Asn Leu 355 360 365 <210> 12 <211> 425 <212> PRT <213> Polyporus brumalis <400> 12 Met Arg Ala Arg Arg Leu Ala Asp Leu Leu Val Leu Gly Ser Thr Tyr 1 5 10 15 Gln Gly Tyr Lys Leu Asp Asp Thr Ala Gly Asn Gln Phe Cys Met Arg 20 25 30 Thr Arg Thr Tyr Lys Glu Ala Gly Thr Thr Ala Ser Lys Ile Gln Asp 35 40 45 Thr Tyr Leu Leu Leu Ser Cys Ser Ser Pro Ser Arg Thr Met Ala Phe 50 55 60 Ala Ile Leu Ala Ser Phe Val Ser Ala Leu Ala Ala Leu Gln Val Ala 65 70 75 80 Asn Gly Ala Leu Thr Arg Arg Val Thr Cys Ala Asp Gly Ser Val Thr 85 90 95 Ser Asn Ala Ala Cys Cys Ala Leu Phe Pro Val Ile Gln Asp Leu Gln 100 105 110 Ala Asn Leu Phe Asp Asn Gly Gly Cys Gly Glu Asp Val His Glu Ser 115 120 125 Leu Arg Leu Thr Phe His Asp Ala Ile Gly Ile Ser Pro Ala Ile Ala 130 135 140 Ala Arg Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ala Leu 145 150 155 160 Phe Glu Asp Ile Glu Thr Asn Phe His Ala Asn Leu Gly Val Asp Glu 165 170 175 Ile Ile Asp Glu Gln Arg Pro Leu Leu Gln Arg Ser Asn Ile Ser Ala 180 185 190 Ala Asp Phe Ile Gln Leu Ala Gly Ala Ile Gly Val Ala Asn Cys Pro 195 200 205 Gly Ala Pro Arg Leu Ala Val Phe Val Gly Arg Gln Asp Ala Thr Gln 210 215 220 Pro Ala Pro Asp Leu Thr Val Pro Glu Pro Phe Asp Thr Val Asp Ser 225 230 235 240 Ile Leu Gln Arg Phe Glu Asp Ala Gly Gly Phe Thr Pro Ala Glu Val 245 250 255 Val Ala Leu Leu Ala Ser His Thr Ile Ala Ala Ala Asp His Val Asp 260 265 270 Pro Ser Ile Pro Gly Thr Pro Phe Asp Ser Thr Pro Glu Leu Phe Asp 275 280 285 Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Thr Leu Phe Pro Gly 290 295 300 Thr Pro Gly Asn Gln Gly Glu Val Glu Ser Pro Val Pro Gly Glu Ile 305 310 315 320 Arg Leu Gln Ser Asp Ser Glu Leu Ala Arg Asp Ser Arg Thr Ala Cys 325 330 335 Glu Trp Gln Ser Phe Val Asn Asn Gln Ala Lys Leu Gln Ser Ala Phe 340 345 350 Ala Ala Ala Phe Arg Lys Met Thr Ile Leu Gly His Asp Glu Ser Glu 355 360 365 Leu Val Asp Cys Ser Asp Val Val Gln Gln Pro Pro Pro Pro Ala Ser 370 375 380 Ala Ala His Phe Pro Ala Gly Leu Ser Asn Ala Asp Ile Glu Gln Ala 385 390 395 400 Cys Ala Ser Thr Pro Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Val 405 410 415 Thr Ser Val Ala Pro Val Pro Pro Ser 420 425 <110> Korea Forest Research Institute <120> Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus          brumalis and expression vector containing specifically <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1316 <212> DNA <213> Polyporus brumalis <400> 1 caccggactc tacggacatg gccttccaag ctctcctctc catagtcgcc gttgttgctg 60 cccttcaagg cgtcgacgct gcgctcaccc gccgcgtcgc ttgcccggac ggcaagaaca 120 ccgcgacgaa cgcggcatgc tgcgccctct tccctatccg ggacgacatc gtcgagaacc 180 tcttccacaa ccagtgcgct gaggaggccc acgagtccct ccgcttgact ttccacgacg 240 ccgtcgcctt ctcgcccgcg gcgcaggccc ggggcgagtt tgtcggcgga ggcgctgacg 300 gctccatcgt cctcttctcc gacatcgaga cgaacttcca tgcgaacgac ggcaccgacg 360 acatcgtcgc gttgcagaag ccgttcatcg ccaagcacaa catctctgcc gctgacttcg 420 tgcaatttgc gggtgccgtc gggctcgctc agtgccccgg tgcacctcag ctcgagttcc 480 tgctcggtcg caaggatgcc actcagcctg ctaatgatgg tcttgttcca gagcccttcg 540 atagcgttga cagcatcctc gctcgcctcc tcgacgtcgg cttccaggac ttcgaggtcg 600 tctggctgct ctccgcccat acggtcgcag ctgccgacag ggtggacccc accatcccgc 660 gcacgccctt cgattctacc ccggagatct tcgacaccca gttcttcatc gagactcagc 720 tccgcgggca gacattcccc ggcgtgggcg gcgagcaggg cgaggtgacg tccccgctcc 780 gcggcgagat gcggctgcaa tcagaccacc tcctcgcacg cgactcgcgc acctcgtgcg 840 agtggcagtc tttcaccaac gaccaggaga agttcgccga gacgttcccg gacgtcatgg 900 gccgccttgc gctcctcggc gtggaccaga gccagctcat cgactgctcg gaagtcatcc 960 ccatcgcgcc gcctctcccc gccagctccc gcccccactt ccccgctggc aagactgctg 1020 ctgatgtcga gcaggcttgc gccgagaccc ccttcccgtc cttccccacc gaccccggcc 1080 cggccacctc tgttgcgccc gttcccaatg catgatcacg ttgtacactc gtacacgacc 1140 cggagtattc gtgttggccc attgtatgct tttcgacttg tggtgtacta catacttagt 1200 ggtatcagga ccgtcgcagc tgcgcccggc gctggttgtg gcggacggtg tacattacct 1260 cgtgtgtgaa acttgtatca ctgtcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1316 <210> 2 <211> 1226 <212> DNA <213> Polyporus brumalis <400> 2 caataggtca acggtcatgg ctttcaaggc tctgctctcc ttcgtctcca tcgtcgccgc 60 catccagggt gccaccgctg cgctcacaag gcgcgtcgct tgccccgacg gcaagaacac 120 tgcgaccaat gcagcgtgct gcgccctctt ccccattcgg gacgacatcg tcaagaacct 180 cttccacaac cagtgcgcag aggaggccca cgagtccctc cgcctcacct tccacgacgc 240 tgtcgcgttc tcgcccgctg cccaagcccg tggccagttc gccggtggag gcgccgacgg 300 ctccatcgtt ctcttctccg aaatcgagac ggccttccac gcaaatggag gcaccgacga 360 gatcgtcgcg ttgcagaagc ccttcatcgc caagcataac atctccgccg ctgacttcgt 420 tcaattcgcg ggcgccgtcg ggctcgctca gtgccccggt gcacctcagc tcgagttcaa 480 gctcggtcgc aaggacgcaa cccgggccgc acctgacggt ctcgtccccg aacctttcga 540 ctcggtcgac aagatccttg cccgcttgct cgacgtcggt ttccaggact tcgaggtcgt 600 atggctgctc tccgcacaca ctgtcgccgc cgccgaccta gtcgacccga cgatcccgcg 660 tacgccattc gactcaactc cggaaatctt cgacacccag tttttcatcg agacccagct 720 ccgcggacag accttccccg gtaagggcgg catccagggc gaggtgacgt ccccgctccg 780 cggcgagatg cgcctgcagt ccgaccatct gctcgcgcgt gactcgcgca catcgtgcga 840 gtggcagtcg ttcaccaacg accaggagaa gtttgcggag acgttcccgg acgtcatggg 900 ccgccttgcg ctcctcggtg tcgacgagag caagctcatt gattgctcgg aggtcattcc 960 gctcgccccg cccctcccgg ccaacatccg cccgcatttc cctgctggaa agacccatgc 1020 cgacatcgag caagcatgtg ctgagacgcc gttcccctcg ggtccggcga ctgtcgtcgc 1080 tcctgttccc aacctttaga ttggtccagg ctctgaaggt catcgttggt cacaggttca 1140 tagacagtgt catggtgctg cagtatatga gtggtcactc gcgaatacta tctgacttat 1200 gatcatcaaa aaaaaaaaaa aaaaaa 1226 <210> 3 <211> 1905 <212> DNA <213> Polyporus brumalis <400> 3 ggcacgaggg ttagacagac atgctgtctg cccgcactgt cttgcaattc cgtatacaac 60 accaacgttc cagggagtgt actctcacca cgaacctcac ctgcccggtg ccggtcctgc 120 ggagcaccca ctgccgtgac tcgggcatcc gctccactgt cgcatcggta ccgtggtcgt 180 actcgcaggc atcggcacca ttcgcacact tgtttggtac cagctcctac gctgcccaca 240 gccgaggtgc cgacaccgtc ctaaatatct ggttctctca ggagcgcacc cgaaccctgg 300 tagaggtgct atgcaagcag cgagagacat gacccgccgg gtgcccgacg caccgtttgg 360 gttaggcagc gacgtgccat tggctgcgtc catattgtac gccgacacgc cgacgtcggc 420 atgccctgta cacccatggc atcgggttat tgcgttcacg ctgtcagaat gggtgcagcg 480 tgattggctg gcctccttgg caagggttta tgtacctttc gcaatttcga ggacatagca 540 gcaccttttg caggatggcg aagagtataa aagagctgtg gcgactgcca ccaaacttca 600 ggacatccac ctttcctcag tcacttcatc tcctcttcgg acaatggcct tcaaaacact 660 cgcgtctttc gtctcggttc ttgcggccct ccaggtcgcc aacggtgccc tcgtacgccg 720 ggtcacttgc gctgacggca gcgtcacgtc caacgcggca tgctgcgcgc tcttcccggt 780 gatccaggat ctccagacaa acctcttcga cggcggcgag tgtggtgagg aggttcacga 840 atcccttcgt ctgaccttcc acgacgccat cggtatctct cccgccatcg cctcccgcgg 900 ccaattcggt ggtggaggtg ccgacggttc gatcgccctc ttcgaagaca tcgagaccaa 960 cttccacgcg aacaacggtg tcgacgagat catcgacgaa cagcgccccc tcatccagcg 1020 ccacaacctc accacggctg acttcatcca gcttgctggt gccatcggtg tcagcaactg 1080 tcctggtgcc cctcgtcttg cagtgttcgt tggtcgcccc gacgccacac agcccgcgcc 1140 cgacctgacc gtcccggagc ccttcgacac cgtcgacagc atcctgcaac gcttcgatga 1200 tgccggtggc ttcactccag cggaggtcgt ggcgctcctc ggctcccaca ccatcgccgc 1260 ggccgaccac gtcgacccgt ccatcccggg aactcccttc gactcgactc ctgagctctt 1320 cgacacccag ttctttatcg agactcagct ccgtggcacg ctcttccccg gcaccggcgg 1380 caaccagggt gaggtcgagt cgcccctccg cggcgagctc cgtctccagt ccgactccga 1440 gctcgcccgt gactctcgca ctgcttgcga gtggcagtcc ttcgtcaaca accaggctaa 1500 gctccagtcc gcgttcgctg ctgccttccg caagatgacg atcctcgggc acgacgaaag 1560 ctcgctgatc gactgttctg acgtcgtccc gactcccccg gccccggcct ccagcgcgca 1620 ccttcccgcc ggcctcaccc acaacgacgt cgagcaggct tgcgccagca cccccttccc 1680 cacgctcccc accgaccccg gtccggccac cactgtcgct cctgtccctc cctcttagag 1740 tgtgtcagac cgtgaagcgg tgctcaggct ggagaacgtc tttcattgtc cgggcgttgc 1800 tggaatgtcg ccacgtatct ttgaccatcg ggtgtatttc tatgtatcat gggctatcgt 1860 agagagtcag attgtcagta aaaaaaaaaa aaaaaaaaaa aaaaa 1905 <210> 4 <211> 2011 <212> DNA <213> Polyporus brumalis <400> 4 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 a 2011 <210> 5 <211> 1502 <212> DNA <213> Polyporus brumalis <400> 5 acgcggggtc agggaggaac gcatcaccga caacagcaaa tggtcgtgtt gtcattggtc 60 acccttcgag tatccgacca tggtgtaacg actcagcgag atacaggacg gtcacgtccg 120 gctcatatgc agagtccgcc agggtataag ggcctcctcc ttcgtccctc cactcctcag 180 cgaactcggc tttcgttacc agaagcacga catgggtttc aaggctctcc tctctattct 240 cgccgtcgtc gcaaggctcg aggtcgccag cgctgcgctt acgcgccgtg ttgcgtgccc 300 cgacgggaag aacaccgcga cgaacgcggc gtgctgcgct ctgttcccga tccgggacga 360 catcgtcgag aacctcttcc acaaccagtg cgccgaggag gcccacgaat ccctccgtct 420 gaccttccac gacgctgtcg cgttctcgcc cgcagctgag gctcgcggcc agttcgccgg 480 tggaggcgcc gacggctcca tcgttctctt ctccgagatc gagacggcat tccacgcaaa 540 cggaggcacc gacgagatcg tcgcgctgca gaagcccttc atcgccaagc acaacatctc 600 ggccgctgac ttcattcaat tcgcgggcgc cgtcgcgctc gctcagtgcc ctggtgcacc 660 tcagctcgag ttcatgctcg gccgcaagga tgctacgcgg gccgcccctg atggtctcgt 720 ccccgagccc ttcgacacaa tcgatgacat cctcgcccgc ttgctcgacg tcgggttcca 780 ggacttcgaa gtcgtgtggc tgctctcagc acacaccgtc gctgccgccg acctggtcga 840 cccgaccatc ccgcgcacgc ccttcgactc gacgccggag atctttgaca cccagttctt 900 catcgagacc cagctccgtg gtctgaagtt cgccggccag ggcggcatcc acggcgaggt 960 caactctccg ctcaccggcg agatgcgcct ccagtcggac cacctcctcg ctcgcgactc 1020 gcgcacggcc tgcgagtggc agtcattcac gaacgaccag gagaagttcg cggagacgtt 1080 cccggatgtg atgggccgcc ttgcgctcct cggtgttgat cagagcacgc tcatcgactg 1140 ctctgaggtc atcccgcttg ccccgcccct gcccgccagc tctcgcccgc acttccccgc 1200 tggcaagacc cacgccgaca tcgaacaggc ttgcgccgac accccgttcc cgaccttccc 1260 cactgaccct ggtccggcca ccaaggtcgc ccccgttccc aacctataag ccgttcaccg 1320 agctggctag cgagcgtcat gaggtccatt tgcgggctac aagagcacac agcaggggtg 1380 ttgctggttg taggtgccgc tggttataga tgctatcgga gcccatgttc cctacagctt 1440 tgaagttatg ttgaataacg aatatcaaac gtttgaattt caaaaaaaaa aaaaaaaaaa 1500 aa 1502 <210> 6 <211> 2245 <212> DNA <213> Polyporus brumalis <400> 6 aagcagtggt aacaacgcag agtacgcggg ataggctcaa gttgtattcc acggccgtgg 60 gccacaaggt tcccgcagca ggacttttac tgctttgtga cgcggccgtg ggagcggtac 120 atctcgggaa gctcaagcta aaatatggcc gacacggcag gatcttaagg ctttgggtcc 180 tgcttcgatc gtgccatctc cgggcctggg acgcatgccc gaggccggag ctcgagctcg 240 aaccacatcg ccgtttctgg tgaccccacc cagagcccct gcgatttgga cctgccaagg 300 gacctacaat cgcacgaacg tcaacttggc gtgtcgtttt cctttttcct tgtgccatag 360 cagtgcagcg gtctgcgcgg tgtacgcctg catgtccgaa gatagagctt cagactcaag 420 ctgtgcactc tcaccaagaa cctctcgctc gccgttgccg aagaccagca tcctggtata 480 tccccatact accgctccac ccgttcgttc cttgacatgg cagccccacc gtggcaatgg 540 agctgtggtt gaggtcgcag gaactgggtg cggcccgcta ccgccttgtt ccatctgcaa 600 tgccgcccgc agccgaggac tcgacgcgcc cgcccatgcc tccgtttctc atgaacgcgc 660 ccttcgcctg gtggacgtgt cgcgtaaata agtccggaac atgtcgctct gggtacccgc 720 tctacggtct tggtcgcccg ccgtgcttca ttggcccgcc gaccatcacg gaccgacaca 780 ccgacgctat ggactaactt ctatatccca tgcgtcggcc agtccattca cgcatgtgaa 840 tgcgcgctcg gcgattggcc gacctcttgg ttttggggtc gacctaccaa gggtataagc 900 tggacgacac tgcaggtaac caattttgca tgcggacgag gacgtataaa gaggctggta 960 cgacggccag caagattcag gacacctacc tcctcctcag ctgttcatct ccttctcgga 1020 caatggcatt cgcaatcctc gcttccttcg tctctgccct cgcggctctt caggtcgcca 1080 acggtgccct tactcgccgg gtgacctgcg ccgacggcag cgtgacgtcc aacgcggcat 1140 gctgcgcgct cttcccggtc attcaggacc tccaggctaa cctcttcgac aacggtggat 1200 gtggcgagga cgtccacgag tcccttcgct tgaccttcca cgacgccatt ggtatatccc 1260 ctgccatcgc cgctcgcggg caattcggtg gcggaggcgc cgacggctcg atcgccctgt 1320 tcgaggacat cgagaccaac ttccacgcga accttggtgt cgatgagatc atcgacgagc 1380 agcgtcccct cctccagcgc tccaacatca gcgcggctga cttcattcag cttgctggtg 1440 cgatcggtgt cgccaactgc cccggtgctc ctcgcctcgc cgtctttgtc ggccgtcagg 1500 atgctactca gcccgcaccc gacttgacag tccctgagcc tttcgacacc gtcgacagca 1560 tcctgcagcg cttcgaggat gccggtggct tcacccccgc tgaagttgtc gccctcctcg 1620 cctctcacac gatcgctgca gccgaccacg tggacccgag catccccgga acgcctttcg 1680 actccacgcc tgagttgttc gacacccagt tcttcatcga gacccagctc cgcggcaccc 1740 tcttccccgg aaccccaggt aaccagggcg aggtcgagtc accagtcccc ggcgagatcc 1800 gtctccagtc tgactctgag ctcgctcgcg actctcgcac tgcttgcgag tggcagtcct 1860 ttgtcaacaa ccaggctaag ctccagtccg cgttcgccgc tgcgttccgc aagatgacga 1920 tcctcggcca cgacgagagc gagctcgtcg actgctccga cgtcgtccag cagcccccgc 1980 ctccggcttc cgctgcgcac ttccccgccg gtctcagcaa cgccgacatc gagcaggcgt 2040 gcgccagcac tcccttcccc actctcccca ccgaccccgg cccggtcacc tcggtcgcac 2100 ctgtccctcc ttcctaaata ttcaccgact agacttgttg gcgctcttga agggttctga 2160 agttctccct gacgtctcgt gtatatatga gtaaacccgt atccaacctg cttctctnta 2220 gcaaaaaaaa aaaaaaaaaa aaaaa 2245 <210> 7 <211> 365 <212> PRT <213> Polyporus brumalis <400> 7 Met Ala Phe Gln Ala Leu Leu Ser Ile Val Ala Val Val Ala Ala Leu   1 5 10 15 Gln Gly Val Asp Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly              20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg          35 40 45 Asp Asp Ile Val Glu Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala      50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro  65 70 75 80 Ala Ala Gln Ala Arg Gly Glu Phe Val Gly Gly Gly Ala Asp Gly Ser                  85 90 95 Ile Val Leu Phe Ser Asp Ile Glu Thr Asn Phe His Ala Asn Asp Gly             100 105 110 Thr Asp Asp Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn         115 120 125 Ile Ser Ala Ala Asp Phe Val Gln Phe Ala Gly Ala Val Gly Leu Ala     130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Leu Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Gln Pro Ala Asn Asp Gly Leu Val Pro Glu Pro Phe Asp Ser                 165 170 175 Val Asp Ser Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe             180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Arg         195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile     210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Gln Thr Phe 225 230 235 240 Pro Gly Val Gly Gly Glu Gln Gly Glu Val Thr Ser Pro Leu Arg Gly                 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr             260 265 270 Ser Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu         275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Gln     290 295 300 Ser Gln Leu Ile Asp Cys Ser Glu Val Ile Pro Ile Ala Pro Pro Leu 305 310 315 320 Pro Ala Ser Ser Arg Pro His Phe Pro Ala Gly Lys Thr Ala Ala Asp                 325 330 335 Val Glu Gln Ala Cys Ala Glu Thr Pro Phe Pro Ser Phe Pro Thr Asp             340 345 350 Pro Gly Pro Ala Thr Ser Val Ala Pro Val Pro Asn Ala         355 360 365 <210> 8 <211> 360 <212> PRT <213> Polyporus brumalis <400> 8 Met Ala Phe Lys Ala Leu Leu Ser Phe Val Ser Ile Val Ala Ala Ile   1 5 10 15 Gln Gly Ala Thr Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly              20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg          35 40 45 Asp Asp Ile Val Lys Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala      50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro  65 70 75 80 Ala Ala Gln Ala Arg Gly Gln Phe Ala Gly Gly Gly Ala Asp Gly Ser                  85 90 95 Ile Val Leu Phe Ser Glu Ile Glu Thr Ala Phe His Ala Asn Gly Gly             100 105 110 Thr Asp Glu Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn         115 120 125 Ile Ser Ala Ala Asp Phe Val Gln Phe Ala Gly Ala Val Gly Leu Ala     130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Lys Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Arg Ala Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Ser                 165 170 175 Val Asp Lys Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe             180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Leu         195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile     210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Gln Thr Phe 225 230 235 240 Pro Gly Lys Gly Gly Ile Gln Gly Glu Val Thr Ser Pro Leu Arg Gly                 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr             260 265 270 Ser Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu         275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Glu     290 295 300 Ser Lys Leu Ile Asp Cys Ser Glu Val Ile Pro Leu Ala Pro Pro Leu 305 310 315 320 Pro Ala Asn Ile Arg Pro His Phe Pro Ala Gly Lys Thr His Ala Asp                 325 330 335 Ile Glu Gln Ala Cys Ala Glu Thr Pro Phe Pro Ser Gly Pro Ala Thr             340 345 350 Val Val Ala Pro Val Pro Asn Leu         355 360 <210> 9 <211> 364 <212> PRT <213> Polyporus brumalis <400> 9 Met Ala Phe Lys Thr Leu Ala Ser Phe Val Ser Val Leu Ala Ala Leu   1 5 10 15 Gln Val Ala Asn Gly Ala Leu Val Arg Arg Val Thr Cys Ala Asp Gly              20 25 30 Ser Val Thr Ser Asn Ala Ala Cys Cys Ala Leu Phe Pro Val Ile Gln          35 40 45 Asp Leu Gln Thr Asn Leu Phe Asp Gly Gly Glu Cys Gly Glu Glu Val      50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Ile Gly Ile Ser Pro  65 70 75 80 Ala Ile Ala Ser Arg Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser                  85 90 95 Ile Ala Leu Phe Glu Asp Ile Glu Thr Asn Phe His Ala Asn Asn Gly             100 105 110 Val Asp Glu Ile Ile Asp Glu Gln Arg Pro Leu Ile Gln Arg His Asn         115 120 125 Leu Thr Thr Ala Asp Phe Ile Gln Leu Ala Gly Ala Ile Gly Val Ser     130 135 140 Asn Cys Pro Gly Ala Pro Arg Leu Ala Val Phe Val Gly Arg Pro Asp 145 150 155 160 Ala Thr Gln Pro Ala Pro Asp Leu Thr Val Pro Glu Pro Phe Asp Thr                 165 170 175 Val Asp Ser Ile Leu Gln Arg Phe Asp Asp Ala Gly Gly Phe Thr Pro             180 185 190 Ala Glu Val Val Ala Leu Leu Gly Ser His Thr Ile Ala Ala Ala Asp         195 200 205 His Val Asp Pro Ser Ile Pro Gly Thr Pro Phe Asp Ser Thr Pro Glu     210 215 220 Leu Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Thr Leu 225 230 235 240 Phe Pro Gly Thr Gly Gly Asn Gln Gly Glu Val Glu Ser Pro Leu Arg                 245 250 255 Gly Glu Leu Arg Leu Gln Ser Asp Ser Glu Leu Ala Arg Asp Ser Arg             260 265 270 Thr Ala Cys Glu Trp Gln Ser Phe Val Asn Asn Gln Ala Lys Leu Gln         275 280 285 Ser Ala Phe Ala Ala Ala Phe Arg Lys Met Thr Ile Leu Gly His Asp     290 295 300 Glu Ser Ser Leu Ile Asp Cys Ser Asp Val Val Pro Thr Pro Pro Ala 305 310 315 320 Pro Ala Ser Ser Ala His Leu Pro Ala Gly Leu Thr His Asn Asp Val                 325 330 335 Glu Gln Ala Cys Ala Ser Thr Pro Phe Pro Thr Leu Pro Thr Asp Pro             340 345 350 Gly Pro Ala Thr Thr Val Ala Pro Val Pro Pro Ser         355 360 <210> 10 <211> 437 <212> PRT <213> Polyporus brumalis <400> 10 Met Pro Gln Thr Pro Arg Glu Ser Gly Ala Cys Val His Ala Val Arg   1 5 10 15 Met Gly Ala Ala Arg Leu Ala Gly Leu Leu Glu His Asp Leu Arg Leu              20 25 30 Ala Arg Ser Arg His Ser Ile Thr Val Lys Ala Leu Ala Asn Glu Tyr          35 40 45 Lys Gly Ala Gly Ala Thr Thr Thr Lys Pro Gln Asp Ile Tyr Leu Ser      50 55 60 Ser Val Ala Pro Ser Pro Leu Arg Thr Met Ala Phe Lys Thr Leu Ala  65 70 75 80 Ser Phe Val Ser Val Leu Ala Ala Phe Gln Ala Ala Asn Gly Ala Leu                  85 90 95 Val Arg Arg Val Thr Cys Ala Asp Gly Ser Val Thr Ala Asn Ala Ala             100 105 110 Cys Cys Ala Leu Phe Pro Val Ile Gln Asp Leu Gln Thr Asn Leu Phe         115 120 125 Asp Gly Gly Glu Cys Gly Glu Glu Val His Glu Ser Leu Arg Leu Thr     130 135 140 Phe His Asp Ala Ile Gly Ile Ser Pro Ala Ile Ala Ser Arg Gly Gln 145 150 155 160 Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ala Ile Phe Asp Asp Ile                 165 170 175 Glu Thr Asn Phe His Ala Asn Asn Gly Val Asp Glu Ile Ile Gly Glu             180 185 190 Gln Arg Pro Leu Ile Gln Arg His Asn Ile Thr Thr Ala Asp Phe Ile         195 200 205 Gln Leu Ala Gly Ala Ile Gly Val Ser Asn Cys Pro Gly Ala Pro Arg     210 215 220 Leu Ala Val Phe Val Gly Arg Pro Asp Ala Thr Gln Pro Ala Pro Asp 225 230 235 240 Leu Thr Val Pro Glu Pro Phe Asp Thr Val Asp Ser Ile Leu Gln Arg                 245 250 255 Phe Asp Asp Ala Gly Gly Phe Thr Pro Glu Glu Val Val Ala Leu Leu             260 265 270 Gly Ser His Thr Ile Ala Ala Ala Asp His Val Asp Pro Ser Ile Pro         275 280 285 Gly Thr Pro Phe Asp Ser Thr Pro Glu Leu Phe Asp Thr Gln Phe Phe     290 295 300 Ile Glu Thr Gln Leu Arg Gly Thr Leu Phe Pro Gly Thr Gly Gly Asn 305 310 315 320 Gln Gly Glu Val Glu Ser Pro Leu Arg Gly Glu Leu Arg Leu Gln Ser                 325 330 335 Asp Ser Glu Leu Ala Arg Asp Ser Arg Thr Ala Cys Glu Trp Gln Ser             340 345 350 Phe Val Asn Asn Gln Ala Lys Leu Gln Ser Ala Phe Ala Ala Ala Phe         355 360 365 Arg Lys Met Thr Ile Leu Gly His Asp Glu Gly Ser Leu Val Asp Cys     370 375 380 Ser Asp Val Val Pro Thr Pro Pro Ala Pro Ala Ser Gln Ala His Leu 385 390 395 400 Pro Ala Gly Leu Thr His Asn Asp Val Glu Gln Ala Cys Ala Ser Thr                 405 410 415 Pro Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Ala Thr Ser Val Ala             420 425 430 Pro Val Pro Pro Ser         435 <210> 11 <211> 365 <212> PRT <213> Polyporus brumalis <400> 11 Met Gly Phe Lys Ala Leu Leu Ser Ile Leu Ala Val Val Ala Arg Leu   1 5 10 15 Glu Val Ala Ser Ala Ala Leu Thr Arg Arg Val Ala Cys Pro Asp Gly              20 25 30 Lys Asn Thr Ala Thr Asn Ala Ala Cys Cys Ala Leu Phe Pro Ile Arg          35 40 45 Asp Asp Ile Val Glu Asn Leu Phe His Asn Gln Cys Ala Glu Glu Ala      50 55 60 His Glu Ser Leu Arg Leu Thr Phe His Asp Ala Val Ala Phe Ser Pro  65 70 75 80 Ala Ala Glu Ala Arg Gly Gln Phe Ala Gly Gly Gly Ala Asp Gly Ser                  85 90 95 Ile Val Leu Phe Ser Glu Ile Glu Thr Ala Phe His Ala Asn Gly Gly             100 105 110 Thr Asp Glu Ile Val Ala Leu Gln Lys Pro Phe Ile Ala Lys His Asn         115 120 125 Ile Ser Ala Ala Asp Phe Ile Gln Phe Ala Gly Ala Val Ala Leu Ala     130 135 140 Gln Cys Pro Gly Ala Pro Gln Leu Glu Phe Met Leu Gly Arg Lys Asp 145 150 155 160 Ala Thr Arg Ala Ala Pro Asp Gly Leu Val Pro Glu Pro Phe Asp Thr                 165 170 175 Ile Asp Asp Ile Leu Ala Arg Leu Leu Asp Val Gly Phe Gln Asp Phe             180 185 190 Glu Val Val Trp Leu Leu Ser Ala His Thr Val Ala Ala Ala Asp Leu         195 200 205 Val Asp Pro Thr Ile Pro Arg Thr Pro Phe Asp Ser Thr Pro Glu Ile     210 215 220 Phe Asp Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Leu Lys Phe 225 230 235 240 Ala Gly Gln Gly Gly Ile His Gly Glu Val Asn Ser Pro Leu Thr Gly                 245 250 255 Glu Met Arg Leu Gln Ser Asp His Leu Leu Ala Arg Asp Ser Arg Thr             260 265 270 Ala Cys Glu Trp Gln Ser Phe Thr Asn Asp Gln Glu Lys Phe Ala Glu         275 280 285 Thr Phe Pro Asp Val Met Gly Arg Leu Ala Leu Leu Gly Val Asp Gln     290 295 300 Ser Thr Leu Ile Asp Cys Ser Glu Val Ile Pro Leu Ala Pro Pro Leu 305 310 315 320 Pro Ala Ser Ser Arg Pro His Phe Pro Ala Gly Lys Thr His Ala Asp                 325 330 335 Ile Glu Gln Ala Cys Ala Asp Thr Pro Phe Pro Thr Phe Pro Thr Asp             340 345 350 Pro Gly Pro Ala Thr Lys Val Ala Pro Val Pro Asn Leu         355 360 365 <210> 12 <211> 425 <212> PRT <213> Polyporus brumalis <400> 12 Met Arg Ala Arg Arg Leu Ala Asp Leu Leu Val Leu Gly Ser Thr Tyr   1 5 10 15 Gln Gly Tyr Lys Leu Asp Asp Thr Ala Gly Asn Gln Phe Cys Met Arg              20 25 30 Thr Arg Thr Tyr Lys Glu Ala Gly Thr Thr Ala Ser Lys Ile Gln Asp          35 40 45 Thr Tyr Leu Leu Leu Ser Cys Ser Ser Pro Ser Arg Thr Met Ala Phe      50 55 60 Ala Ile Leu Ala Ser Phe Val Ser Ala Leu Ala Ala Leu Gln Val Ala  65 70 75 80 Asn Gly Ala Leu Thr Arg Arg Val Thr Cys Ala Asp Gly Ser Val Thr                  85 90 95 Ser Asn Ala Ala Cys Cys Ala Leu Phe Pro Val Ile Gln Asp Leu Gln             100 105 110 Ala Asn Leu Phe Asp Asn Gly Gly Cys Gly Glu Asp Val His Glu Ser         115 120 125 Leu Arg Leu Thr Phe His Asp Ala Ile Gly Ile Ser Pro Ala Ile Ala     130 135 140 Ala Arg Gly Gln Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Ala Leu 145 150 155 160 Phe Glu Asp Ile Glu Thr Asn Phe His Ala Asn Leu Gly Val Asp Glu                 165 170 175 Ile Ile Asp Glu Gln Arg Pro Leu Leu Gln Arg Ser Asn Ile Ser Ala             180 185 190 Ala Asp Phe Ile Gln Leu Ala Gly Ala Ile Gly Val Ala Asn Cys Pro         195 200 205 Gly Ala Pro Arg Leu Ala Val Phe Val Gly Arg Gln Asp Ala Thr Gln     210 215 220 Pro Ala Pro Asp Leu Thr Val Pro Glu Pro Phe Asp Thr Val Asp Ser 225 230 235 240 Ile Leu Gln Arg Phe Glu Asp Ala Gly Gly Phe Thr Pro Ala Glu Val                 245 250 255 Val Ala Leu Leu Ala Ser His Thr Ile Ala Ala Ala Asp His Val Asp             260 265 270 Pro Ser Ile Pro Gly Thr Pro Phe Asp Ser Thr Pro Glu Leu Phe Asp         275 280 285 Thr Gln Phe Phe Ile Glu Thr Gln Leu Arg Gly Thr Leu Phe Pro Gly     290 295 300 Thr Pro Gly Asn Gln Gly Glu Val Glu Ser Pro Val Pro Gly Glu Ile 305 310 315 320 Arg Leu Gln Ser Asp Ser Glu Leu Ala Arg Asp Ser Arg Thr Ala Cys                 325 330 335 Glu Trp Gln Ser Phe Val Asn Asn Gln Ala Lys Leu Gln Ser Ala Phe             340 345 350 Ala Ala Ala Phe Arg Lys Met Thr Ile Leu Gly His Asp Glu Ser Glu         355 360 365 Leu Val Asp Cys Ser Asp Val Val Gln Gln Pro Pro Pro Ala Ser     370 375 380 Ala Ala His Phe Pro Ala Gly Leu Ser Asn Ala Asp Ile Glu Gln Ala 385 390 395 400 Cys Ala Ser Thr Pro Phe Pro Thr Leu Pro Thr Asp Pro Gly Pro Val                 405 410 415 Thr Ser Val Ala Pro Val Pro Pro Ser             420 425  

Claims (5)

서열 1, 서열 2, 서열 3, 서열 4, 서열 5 및 서열 6 중에서 선택된 어느 하나의 염기서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 유전자. Polyporus brumalis manganese peroxidase gene consisting of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 서열 7, 서열 8, 서열 9, 서열 10, 서열 11 및 서열 12 중에서 선택된 어느 하나의 아미노산 서열로 이루어진 겨울우산버섯(Polyporus brumalis) 망간에이즈 퍼옥시다아제(manganese peroxidase) 효소.SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: consisting of any one amino acid sequence selected from SEQ ID NO: 11 and 12 winter umbrella mushroom (Polyporus brumalis ) manganese peroxidase enzyme. 제1항의 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터.A recombinant vector containing the manganese peroxidase gene of claim 1. 제3항의 망간에이즈 퍼옥시다아제 유전자를 함유하는 재조합 벡터로 형질전환된 대장균(E. coli). E. coli transformed with the recombinant vector containing the manganese peroxidase gene of claim 3. 제1항의 망간에이즈 퍼옥시다아제 유전자를 함유하는 리그닌 분해 기능성이 강화된 겨울우산버섯.Winter umbrella mushrooms with enhanced lignin-degrading functionality containing the manganese peroxidase gene of claim 1.
KR1020070082453A 2007-08-16 2007-08-16 Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof KR100888481B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020070082453A KR100888481B1 (en) 2007-08-16 2007-08-16 Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020070082453A KR100888481B1 (en) 2007-08-16 2007-08-16 Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof

Publications (2)

Publication Number Publication Date
KR20090017925A KR20090017925A (en) 2009-02-19
KR100888481B1 true KR100888481B1 (en) 2009-03-16

Family

ID=40686463

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020070082453A KR100888481B1 (en) 2007-08-16 2007-08-16 Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof

Country Status (1)

Country Link
KR (1) KR100888481B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111808831B (en) * 2020-07-13 2022-09-02 浙江康星生物科技有限公司 Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0975079A (en) * 1995-09-12 1997-03-25 Kobe Steel Ltd Manganese peroxidase, manganese peroxidase dna, recombinant vector containing it and its transformant
US6642439B2 (en) 2000-07-25 2003-11-04 Agency Of Industrial Science And Technology Basidiomycete manganese peroxidase gene-transferred plant and a method for removing an environmental contaminant using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0975079A (en) * 1995-09-12 1997-03-25 Kobe Steel Ltd Manganese peroxidase, manganese peroxidase dna, recombinant vector containing it and its transformant
US6642439B2 (en) 2000-07-25 2003-11-04 Agency Of Industrial Science And Technology Basidiomycete manganese peroxidase gene-transferred plant and a method for removing an environmental contaminant using the same

Also Published As

Publication number Publication date
KR20090017925A (en) 2009-02-19

Similar Documents

Publication Publication Date Title
Zhang et al. Cloning and functional analysis of a laccase gene during fruiting body formation in Hypsizygus marmoreus
Cullen Recent advances on the molecular genetics of ligninolytic fungi
Hildén et al. The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent
Galhaup et al. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions
Hoshida et al. Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts
Lankinen et al. Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization
Pozdnyakova et al. Yellow laccase from the fungus Pleurotus ostreatus D1: purification and characterization
Otte et al. Characterization and oxidative in vitro cross-linking of an extensin-like protein and a proline-rich protein purified from chickpea cell walls
Andrawis et al. Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium
Boldo et al. Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae
Judelson et al. Structure and expression of a gene encoding heat-shock protein Hsp70 from the Oomycete fungus Bremia lactucae
Janusz et al. Cloning and characterization of a laccase gene from biotechnologically important basidiomycete Cerrena unicolor
Irie et al. Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycete Pleurotus ostreatus
KR100888481B1 (en) Manganese peroxidase cDNAs from basidiomycete fungus, Polyporus brumalis and expression vector containing thereof
Hilden et al. Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata
Maeda et al. Manganese peroxidase gene of the perennial mushroom Elfvingia applanata: cloning and evaluation of its relationship with lignin degradation
Kim et al. Genome-wide identification and characterization of novel laccase genes in the white-rot fungus Flammulina velutipes
Menaka et al. Cloning of laccase gene from a newly isolated 2, 4-dichlorophenol degrading Bacillus subtilis from dyeing industry sites
Ryu et al. Molecular characteristics of two laccase from the basidiomycete fungus Polyporus brumalis
CN110494568A (en) Use the continuous glucose monitoring of FAD dependent glucose dehydrogenase
CN109715799A (en) Modified sarcosine oxidase and its gene and manufacturing method
Mönkemann et al. Components of the ligninolytic system of Fusarium oxysporum and Trichoderma atroviride
Hamann et al. Discovery, cloning and heterologous expression of secreted potato proteins reveal erroneous pre-mRNA splicing in Aspergillus oryzae
Yakovlev et al. Identification and analysis of differentially expressed Heterobasidion parviporum genes during natural colonization of Norway spruce stems
Ishfaq et al. Biochemical and molecular studies of various enzymes activity in fungi

Legal Events

Date Code Title Description
A201 Request for examination
E701 Decision to grant or registration of patent right
GRNT Written decision to grant