KR20180037424A - 재조합 단백질, 재조합 미생물 및 그를 이용한 고분자 물질을 분해하는 방법 - Google Patents
재조합 단백질, 재조합 미생물 및 그를 이용한 고분자 물질을 분해하는 방법 Download PDFInfo
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- KR20180037424A KR20180037424A KR1020160127547A KR20160127547A KR20180037424A KR 20180037424 A KR20180037424 A KR 20180037424A KR 1020160127547 A KR1020160127547 A KR 1020160127547A KR 20160127547 A KR20160127547 A KR 20160127547A KR 20180037424 A KR20180037424 A KR 20180037424A
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- Prior art keywords
- leu
- ile
- ala
- lys
- glu
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Abstract
재조합 단백질, 상기 재조합 단백질을 코딩하는 유전자, 상기 유전자를 포함하는 재조합 미생물, 및 그를 이용하여 고분자 물질을 분해하는 방법에 관한 것이다.
Description
재조합 단백질, 상기 재조합 단백질을 코딩하는 유전자, 재조합 미생물, 및 그를 이용하여 고분자 물질을 분해하는 방법에 관한 것이다.
물성이 다양하고, 가공이 용이한 플라스틱이라 불리는 고분자 물질은 계속되는 연구에 의하여 점차 유리, 금속, 종이, 목재, 석재 등과 같은 기존의 재료들을 대체하여 산업용 재료로부터 1회용 소모품에 이르기까지 다양한 용도로 사용되고 있고, 그 사용량이 증가되고 있다. 그러나, 사용된 플라스틱 폐기물의 처리는 소각 또는 매립에 의존하고 있는 실정이고, 환경호르몬 누출과 맹독성 다이옥신 검출 폐기물의 불완전 연소에 의하여 토양, 하천, 대기 등의 심각한 환경오염 문제를 야기시키고 있다. 이러한 문제를 해결하기 위해 생분해성 또는 생붕괴성 플라스틱의 실용화 및 사용 의무화의 압력이 거세지면서 미국, 일본, 독일, 이탈리아 등 다른 국가에서는 다양한 플라스틱 제품에 생분해성 고분자 사용을 의무화하는 등 생분해성 플라스틱의 실용화가 활발히 진행되고 있다. 현재, 산업적으로 가장 많이 사용되는 합성 플라스틱은 폴리에틸렌 (polyethylene : PE), 폴리우레탄 (polyurethane : PUR), 나일론 (nylon), 폴리비닐 알콜 (polyvinylalcohol : PVA) 등이다. 이와 같은 합성 플라스틱의 분해 촉진 방법은 광분해, 열분해, 생분해 등과 이러한 방법들을 복합적으로 사용하는 복합분해가 있다. 광분해는 태양광의 자외선 즉 295 nm 내지 400 nm의 파장의 빛을 이용하여 고분자의 산화를 유도하고, 열분해는 고분자에 고온의 열을 가하여 물성을 변화시킨다. 마지막으로 생분해는 고분자가 호기적 또는 혐기적으로 미생물에 의해 분해되는 것이다. 이러한 분해 방법들 중에서 가장 효율이 높은 것은 미생물에 의한 생분해이고, 최근 광 및 열분해는 생분해를 수행하기 전 생분해를 촉진하기 위한 전처리의 개념으로 이용되는 추세여서, 미생물에 의한 분해 방법에 관한 연구가 지속되고 있다.
일 양상은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질 또는 이들의 조합을 제공한다.
다른 양상은 상기 재조합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 유전자를 제공한다.
다른 양상은 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물을 제공한다.
다른 양상은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 미생물, 그 분해물 (lysate) 또는 그 분해물의 분획을 고분자 물질과 접촉시켜 상기 고분자 물질을 분해하는 단계를 포함하는, 고분자 물질을 분해하는 방법을 제공한다.
본 명세서에서 사용된 용어 "유전자 (gene)"는 특정 단백질 또는 폴리펩티드를 발현하는 핵산 단편을 의미하며, 코딩영역 또는 코딩영역 외 5'-비코딩 서열 (5'-non coding 서열)과 3'-비코딩 서열 (3'-non coding 서열) 등의 조절 (regulatory) 서열을 포함할 수 있다. 상기 조절 영역은 프로모터, 인핸서, 오퍼레이터, 리보좀 결합 부위, polyA 결합 서열, 터미네이터 영역 등을 포함할 수 있다.
본 발명의 폴리뉴클레오티드 또는 폴리펩티드의 "서열 동일성 (sequence identity)"은 특정 비교 영역에서 양 서열을 최대한 일치되도록 얼라인시킨 후 서열간의 염기 또는 아미노산 잔기의 동일한 정도를 의미한다. 서열 동일성은 특정 비교 영역에서 2개의 서열을 최적으로 얼라인하여 비교함으로써 측정되는 값으로서, 비교 영역 내에서 서열의 일부는 대조 서열 (reference sequence)과 비교하여 부가 또는 삭제되어 있을 수 있다. 서열 동일성 백분율은 예를 들면, 비교 영역 전체에서 두 개의 최적으로 정렬된 서열을 비교하는 단계, 두 서열 모두에서 동일한 아미노산 또는 뉴클레오티드가 나타나는 위치의 갯수를 결정하여 일치된 (matched) 위치의 갯수를 수득하는 단계, 상기 일치된 위치의 갯수를 비교 범위 내의 위치의 총 갯수 즉, 범위 크기로 나누는 단계, 및 상기 결과에 100을 곱하여 서열 동일성의 백분율을 수득하는 단계에 의해 계산될 수 있다. 상기 서열 동일성의 퍼센트는 공지의 서열 비교 프로그램을 사용하여 결정될 수 있으며, 상기 프로그램의 일례로 BLASTN 또는 BLASTP (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc) 등을 들 수 있다.
여러 종의 동일하거나 유사한 기능이나 활성을 가지는 폴리펩티드 또는 폴리뉴클레오티드를 확인하는데 있어서 여러 수준의 서열 동일성을 사용할 수 있다. 예를 들어, 50%이상, 55%이상, 60%이상, 65%이상, 70%이상, 75%이상, 80%이상, 85%이상, 90%이상, 95%이상, 96%이상, 97%이상, 98%이상, 99%이상 또는 100% 등을 포함하는 서열 동일성이다.
본 명세서에서 사용된 용어 "활성 증가 (increase in activity)", "증가된 활성 (increased activity)", 또는 "증가된 수준 (increased level)"은 세포, 폴리펩티드, 단백질, 또는 효소의 활성의 검출가능한 증가를 나타낼 수 있다. "활성 증가 (increase in activity)", "증가된 활성 (increased activity)", 또는 "증가된 수준 (increased level)"은 주어진 유전적 변형 (genetic modification)을 갖지 않은 세포, 폴리펩티드, 단백질, 또는 효소 (예를 들면, 본래 또는 "야생형 (wild-type)"의 세포, 폴리펩티드, 단백질, 또는 효소)와 같은, 동일한 타입의 비교 세포, 폴리펩티드, 단백질, 또는 효소의 수준보다 더 높은 변형된 (예를 들면, 유전적으로 조작된) 세포, 폴리펩티드, 단백질, 또는 효소의 활성을 나타낼 수 있다. "세포의 활성 (cell activity)"이란 세포의 특정 폴리펩티드, 단백질 또는 효소의 활성을 나타낼 수 있다. 예를 들면, 상기 변형된 또는 조작된 세포, 폴리펩티드, 단백질, 또는 효소의 활성은 동일 타입의 조작되지 않은 세포, 폴리펩티드, 단백질, 또는 효소, 예를 들면, 야생형 세포, 폴리펩티드, 단백질, 또는 효소의 활성보다 약 5% 이상, 약 10% 이상, 약 15% 이상, 약 20% 이상, 약 30% 이상, 약 50% 이상, 약 60% 이상, 약 70% 이상, 또는 약 100% 이상 증가된 것일 수 있다. 세포 중 특정 폴리펩티드, 단백질 또는 효소의 활성은 모세포, 예를 들면, 조작되지 않은 세포 중의 동일 폴리펩티드, 단백질 또는 효소의 활성보다 약 5% 이상, 약 10% 이상, 약 15% 이상, 약 20% 이상, 약 30% 이상, 약 50% 이상, 약 60% 이상, 약 70% 이상, 또는 약 100% 이상 증가된 것일 수 있다. 폴리펩티드, 단백질 또는 효소의 증가된 활성을 갖는 세포는 당업계에 공지된 임의의 방법을 사용하여 확인될 수 있다. 상기 증가된 활성을 갖는 세포는, 유전적 변형을 갖지 않은 세포에 비하여 하나 이상의 효소 또는 폴리펩티드의 활성을 증가시키는 유전적 변형을 갖는 것일 수 있다.
일 양상은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질 또는 이들의 조합을 제공한다.
상기 재조합 단백질은, 서열번호 1의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 서열번호 2의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 서열번호 3의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 서열번호 4의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 서열번호 5의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 서열번호 6의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질, 및 서열번호 7의 아미노산 서열과 95% 이상, 96% 이상, 97% 이상, 98% 이상, 또는 99% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질로 이루어진 군으로부터 선택되는 1종 또는 2종 이상의 재조합 단백질을 포함한다. 또한, 상기 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질은, 각각 서열번호 1,2,3,4,5,6, 및 7의 아미노산 서열을 포함하는 단백질과 각각 그 기능이 동일하면서, 하나 이상의 아미노산이 치환, 결실, 삽입 또는 이들 조합에 의해 변형된 것을 포함한다.
상기 재조합 단백질은 옥시게나제 (oxygenase) 활성을 갖는 것일 수 있다. 옥시게나제는 산소 원자가 기질에 결합하는 산화 반응을 촉매하는 효소를 의미하며, 예를 들면 고분자 물질에 카르보닐기 (carbonyl)를 도입할 수 있거나, 고분자 물질에 탄소-산소 결합를 도입할 수 있거나, 또는 고분자 물질의 탄소-수소 결합 또는 탄소-탄소 결합을 산화시킬 수 있는 효소이다. 상기 재조합 단백질은 고분자 물질의 탄소와 수소 사이의 결합 또는 탄소와 탄소 사이의 결합에 작용하는 것일 수 있다. 즉 상기 재조합 단백질은 고분자 물질의 탄소-수소 결합 또는 탄소-탄소 결합을 탄소-산소 결합, 즉 카르보닐기 (carbonyl group)로 전환하는 것을 촉매할 수 있다.
상기 아미노산 서열을 갖는 단백질은 바실러스 (Bacillus) 속 미생물 유래의 것일 수 있다. 상기 바실러스 속 미생물은 바실러스 속 YP1일 수 있다.
다른 양상은 상기 재조합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 유전자를 제공한다. 상기 유전자는 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열을 포함할 수 있다.
또는, 상기 뉴클레오티드 서열을 포함하는 폴리뉴클레오티드는 숙주 세포인 재조합 미생물에 대하여 코돈 최적화된 것일 수 있다. 상기 코돈 최적화란 동일한 아미노산을 암호화하지만 내재적 코돈 중 하나 이상이 해당 숙주에서 발현에 유리한 코돈으로 치환된 유전자를 생성하는 것을 나타낸다.
상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 어느 하나의 뉴클레오티드 서열을 포함하는 것일 수 있다. 서열번호 1의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 8의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 2의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 9의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 3의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 10의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 4의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 11의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 5의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 12의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 6의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 13의 뉴클레오티드 서열을 포함할 수 있다. 서열번호 7의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열은 서열번호 14의 뉴클레오티드 서열을 포함할 수 있다.
다른 양상은 상기 유전자를 포함하는 발현 벡터를 제공한다. 상기 유전자는 벡터 중에 포함된 것일 수 있다. 상기 벡터는 폴리뉴클레오티드 또는 유전자를 미생물 내로 도입하는데 사용될 수 있는 것이면 어느 것이나 포함된다. 상기 유전자는 삽입되는 벡터와 공동으로 작용하는 제한 효소 작용 부위를 포함하고 있어 효과적으로 발현 벡터에 클로닝될 수 있다. 상기 발현 벡터는 상기 재조합 단백질을 발현시킬 수 있도록 통상의 프로모터 서열, 번역개시 서열 및 터미네이터 서열이 포함될 수 있다. 상기 벡터는 플라스미드 또는 바이러스 벡터일 수 있다.
다른 양상은 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물을 제공한다.
상기 재조합 미생물은 모균주에 비하여 증가된 옥시게나제 활성을 갖는 것일 수 있다. 상기 재조합 미생물은 산화 반응을 촉매하는 단백질을 생산하는 재조합 미생물로서, 상기 산화 반응에 의하여 고분자 물질의 결합을 분해시키는 재조합 단백질을 생산할 수 있도록 유전적 변형 (genetic modification)을 갖는 것일 수 있다. 상기 유전적 변형은 외래 유전자 또는 외래 뉴클레오티드의 도입을 포함한다.
상기 재조합 미생물은 모균주에 비하여 증가된 카피 수의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를 포함하는 것일 수 있다.
상기 재조합 미생물은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를, 하나 이상, 예를 들면, 2 이상, 3 이상, 3 이상, 3 이상, 4 이상, 5 이상, 10 이상, 또는 50 이상을 포함할 수 있다. 상기 재조합 미생물에 복수 개의 유전자가 포함되는 경우, 이들은 서로 동일한 또는 다른 유전자일 수 있다. 상기 유전자는 상기 미생물의 게놈에 통합되거나, 그와 독립적으로 존재하는 것일 수 있다.
상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 하나 이상의 뉴클레오티드 서열을 포함하는 것일 수 있다. 서열번호 1 내지 7의 아미노산 서열, 서열번호 8 내지 14의 뉴클레오티드 서열 및 재조합 단백질에 대하여는 상기한 바와 같다.
상기 재조합 미생물에 있어서, 상기 유전자는 당업계에 알려진 통상적인 방법, 예를 들면, 형질전환, 전기천공 등의 방법에 의하여 상기 미생물 내로 도입될 수 있다.
상기 재조합 미생물은 박테리아, 효모 (yeast) 또는 진균 (fungi)일 수 있다. 상기 박테리아는 그람 양성 또는 그람 음성 박테리아일 수 있다. 상기 그람 음성 박테리아는 에스케리키아 (Escherichia) 속, 잔토모나스 (Xanthomonas) 속, 살모넬라 (Samonellar) 속, 또는 슈도모나스 (Pseudomonas) 속에 속하는 것일 수 있다. 상기 에스케리키아 속 미생물은 대장균일 수 있다. 그람 양성 박테리아는 코리네박테리움 (Corynebacterium) 속, 또는 바실러스 (Bacillus) 속에 속하는 것일 수 있다. 상기 단백질을 재조합 미생물을 이용하여 다량으로 생산할 수 있다. 유전공학적 방법은 유전자 조작에 의해 상기 단백질을 박테리아 또는 진균 등의 숙주 세포에서 다량으로 생산하는 방법이다.
다른 양상은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 미생물, 그 분해물 (lysate) 또는 그 분해물의 분획을 포함하는, 고분자 물질을 분해하기 위한 조성물을 제공한다.
상기 미생물은 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물인 것일 수 있다.
상기 재조합 미생물은 모균주에 비하여 증가된 카피수의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를 포함하는 것일 수 있다. 상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 하나 이상의 뉴클레오티드 서열을 갖는 것일 수 있다.
상기 조성물은 상기 고분자 물질을 분해시키는 것일 수 있다. 상기 조성물은 상기 고분자 물질의 분해를 촉진시킬 수 있다. 상기 분해는, 산화 반응, 또는 고분자 물질의 탄소-수소 결합 또는 탄소-탄소 결합을, 탄소-산소 결합, 즉 카르보닐기 (carbonyl group)로 전환하는 것을 촉매하는 것일 수 있다. 또한, 상기 분해는, 고분자 물질을 상기 고분자 물질의 반복 단위에 가깝도록 나누는 것, 또는 고분자 물질이 파단할 때까지의 가해지는 힘 또는 강도가 감소하는 것을 의미할 수 있다. 상기 파단할 때까지의 가해지는 힘 또는 강도는 인장강도로 측정할 수 있다. 상기 인장강도는 통상의 기술자에게 알려진 방법에 의해 측정될 수 있다. 상기 인장강도는 예를 들면 Text Analyzer, 만능강도시험기 또는 사출기를 통해 측정할 수 있다. 또한, 상기 분해는 분자량, 질량, 무게, 결정도, 결정층 두께, 융해 온도, 결정의 평균 크기의 감소, 또는 작은 결정의 함량 증가를 의미할 수 있다.
본 명세서에서 사용된 용어 "고분자 (macromolecule)"는 중합체 (polymer) 또는 공중합체 (copolymer)를 포함하는 것을 의미한다.
본 명세서에서 사용된 용어 "계"는 고분자 자체 또는 이의 유도체를 포함하는 것을 의미한다.
상기 고분자 물질은 폴리알킬렌계 고분자, 폴리아크릴계 고분자, 및 폴리스티렌계 고분자로부터 선택된 1종 이상의 고분자 물질을 포함하는 것일 수 있다.
상기 고분자 물질의 중량평균분자량 (Mw)은 약 10,000 내지 약 300,000Da, 약 50,000 내지 약 250,000Da, 약 100,000 내지 약 200,000Da, 또는 약 130,000 내지 약 180,000Da일 수 있다. 상기 폴리알킬렌계 고분자는 탄소수 1 내지 10의 알킬렌 반복 단위 (repeat unit)를 포함할 수 있다. 예를 들면, 상기 폴리알킬렌계 고분자의 알킬렌 반복 단위의 탄소수는 2 내지 8, 2 내지 6, 또는 2 내지 4일 수 있다. 상기 반복단위는 에틸렌, 프로필렌, 또는 부틸렌일 수 있다. 상기 폴리알킬렌계 고분자는 예를 들면 폴리에틸렌, 폴리프로필렌, 또는 폴리부틸렌일 수 있다. 폴리에틸렌은 매우 안정하고, 에틸렌 단량체들의 중합반응에 의하여 합성되는 고분자 물질이다. 또한, 폴리에틸렌은 안정한 탄소-탄소 및 탄소-수소 결합, 높은 분자량, 산화 및 가수분해가 가능한 결합의 부재, 높은 소수성 때문에 분해가 어려운 난점이 있었다. 그러나, 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질 또는 이들의 조합은 다른 UV 분해, 열분해, 광분해, 또는 강산과 같은 화학분해 없이도, 고분자 물질을 현저하게 생분해할 수 있다.
서열번호 1 내지 7의 아미노산 서열, 서열번호 8 내지 14의 뉴클레오티드 서열, 재조합 단백질, 및 재조합 미생물에 대하여는 상기한 바와 같다.
다른 양상은 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 미생물, 그 분해물 (lysate) 또는 그 분해물의 분획을 고분자 물질과 접촉시켜 상기 고분자 물질을 분해하는 단계를 포함하는, 고분자 물질을 분해하는 방법을 제공한다.
상기 미생물은 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물인 것일 수 있다. 상기 재조합 미생물은 모균주에 비하여 증가된 카피수의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를 포함하는 것일 수 있다. 상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 하나 이상의 뉴클레오티드 서열을 포함하는 것일 수 있다.
상기 분해물은 상기 미생물을 파괴하여 그 내용물이 세포 외부로 노출되도록 한 상태를 말한다. 상기 분해물은, 효소, 열, 압력 등에 의하여 세포를 파괴하여 얻어질 수 있다. 상기 분획은 수성 용매 또는 유성 용매에 용해되는 물질로부터 분리하여 취하여진 것일 수 있다.
상기 접촉은 액체 또는 고체상에서 이루어지는 것일 수 있다. 상기 접촉은 예를 들면 배지 중에 배양되는 미생물, 미생물의 배양물, 그들의 분해물, 또는 그들의 분해물의 분획과 상기 고분자 물질을 접촉시킴으로써 이루어질 수 있다.
상기 접촉은 고분자 물질과, 상기 미생물, 미생물의 배양물, 그들의 분해물, 또는 그들의 분해물의 분획을 접촉시키는 액체-액체 접촉일 수 있다. 상기 액체-액체 접촉은 혼합하는 것을 포함한다. 상기 접촉은 밀폐된 용기 또는 개방된 용기 중에서 수행되는 것일 수 있다. 상기 접촉은 혐기 조건 또는 호기 조건에서 수행되는 것일 수 있다. 상기 접촉은 상기 미생물의 생육 단계가 지수기 (exponential phase), 또는 정체기 (stationary phase)인 때에 이루어지는 것일 수 있다.
상기 접촉은 상기 미생물을 상기 고분자 물질을 함유하는 시료와 접촉시키면서 배양 또는 인큐베이션하는 단계를 포함하는 것일 수 있다. 상기 접촉은 밀폐된 용기 중에서 미생물이 생존 가능한 조건에서 수행되는 것일 수 있다. 상기 생존 가능한 조건은 미생물이 증식하는 조건 또는 휴지 상태(resting sate)로 있게 하는 조건일 수 있다. 상기 배양은 미생물을 증식하는 조건하에서 이루어질 수 있다. 상기 배양은 상기 미생물을 배양하기 위한 조건하에 배양하는 것을 의미할 수 있다. 이러한 배양하기 위한 조건은 예를 들면, 상기 균주가 이용하는 탄소원, 질소원 또는 산소 조건일 수 있다. 상기 탄소원은 단당류, 이당류 또는 다당류가 포함된다. 상기 탄소원은 글루코스, 프럭토스, 수크로스, 만니톨, 만노스, 말토스, 락토스, 자일로스, 글리세롤, 소르비톨, 셀로비오스, 에탄올 또는 갈락토스일 수 있다. 상기 미생물인 이용할 수 있는 질소원은 유기 질소 화합물, 또는 무기 질소 화합물일 수 있다. 질소원의 예는 아미노산, 아미드, 아민, 질산염, 또는 암모늄염일 수 있다. 상기 미생물 배양에 사용되는 배지는 적절한 보충물을 함유한 최소 또는 복합 배지와 같은, 숙주 세포의 성장에 적합한 임의의 통상적인 배지일 수 있다. 적합한 배지는 상업적인 판매자로부터 입수 가능하고 또는 공지된 제조법에 따라 제조될 수 있다. 상기 배양에 사용되는 배지는 상기 미생물의 요구조건을 만족시킬 수 있는 배지일 수 있다. 상기 배지는 탄소원, 질소원, 염, 미량 원소, 및 이들의 조합으로 이루어진 군으로부터 선택되는 배지일 수 있다. 상기 배양은 호기성 조건에서 배양하는 것일 수 있다. 배양 온도는 약 15 내지 35 ℃일 수 있다. 배양 배지의 pH는 약 4 내지 약 8일 수 있다. 상기 배양은 정치배양 (static culture)이거나 교반하 배양일 수 있다.
상기 접촉은 배치(batch) 또는 연속적으로 수행될 수 있다. 상기 접촉은 예를 들면, 상기 분해시키는 단계에서 얻어진 접촉된 고분자 물질을 신선한 옥시게나제 활성을 갖는 단백질을 코딩하는 외래 유전자를 포함하는 재조합 미생물로서, 상기 재조합 미생물은 모균주에 비하여 증가된 옥시게나제 활성을 갖는 것인 재조합 미생물, 그 분해물 또는 그 분해물의 분획과 접촉시키는 단계를 포함할 수 있다.
이러한 재조합 미생물, 그 분해물 또는 그 분해물의 분획과의 접촉은 1회, 2회 이상, 예를 들면, 2, 3, 5, 또는 10 회 이상 이루어질 수 있다. 상기 접촉은 고분자 물질이 원하는 반복 단위에 도달할 때까지 지속되거나, 또는 반복될 수 있다. 상기 접촉은 1 내지 15일, 2 내지 14일, 3 내지 12일, 4 내지 10일, 5 내지 9일, 또는 6 내지 8일 동안 수행되는 것일 수 있다.
상기 방법에 있어서, 상기 고분자 물질은 1회 접촉에 대하여 1 내지 500mg, 2 내지 250mg, 5 내지 100mg, 10 내지 50mg, 20 내지 40mg, 또는 25 내지 35mg을 분해 대상으로 적용할 수 있다.
상기 방법에 있어서, 상기 접촉은 pH 4 내지 10, pH 5 내지 9, pH 6 내지 8, 또는 pH 7 내지 8의 조건으로 수행되는 것일 수 있다.
상기 미생물, 그 분해물 (lysate) 또는 그 분해물의 분획 중에는 10 내지 200mg, 15 내지 150mg, 16 내지 120mg, 또는 17 내지 110mg의 재조합 단백질이 포함되어 있을 수 있다.
서열번호 1 내지 7의 아미노산 서열, 서열번호 8 내지 14의 뉴클레오티드 서열, 재조합 단백질, 분해, 고분자 물질에 대하여는 상기한 바와 같다.
다른 양상에 따른 재조합 미생물은 고분자 물질을 제거하는데 사용될 수 있다. 용어 "제거"는 상기 고분자 물질의 양을 감소시키는 것을 포함한다. 상기 감소는 완전하게 제거하는 것을 포함한다.
상기 재조합 미생물을 이용하여, 합성 고분자 물질의 생분해를 촉진함으로써, 친환경적으로 합성 고분자 물질을 폐기할 수 있다.
도 1은 도 1은 pET28a-PE의 벡터 지도를 나타낸다.
도 2는 T2, T4, T5, T8, T9 및 T10 단백질을 포함하는 조추출물이 폴리에틸렌의 인장강도를 감소시킨 결과를 확인한 그래프이다.
도 3은 T4, T6, T8 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다.
도 4는 T9 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다.
도 2는 T2, T4, T5, T8, T9 및 T10 단백질을 포함하는 조추출물이 폴리에틸렌의 인장강도를 감소시킨 결과를 확인한 그래프이다.
도 3은 T4, T6, T8 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다.
도 4는 T9 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실시예
1: 서열번호 1 내지 7의 폴리펩티드를 발현하는 재조합 대장균 및 그를 이용한 고분자 물질의 분해
본 실시예에서는 서열번호 1 내지 7의 폴리펩티드를 발현하는 재조합 대장균을 제작하고, 그를 이용하여 폴리에틸렌의 분해 효과를 확인하였다.
(1) 서열번호 1 내지 7의 폴리펩티드를 발현하는 재조합 대장균의 제작
Bacillus sp. YP1 유래 유전자는 모두 대장균의 코돈에 최적화되도록 DNA를 합성하여 제작하였다 (Cosmogenetech). 이 DNA를 주형으로 하고, 서열번호 15 및 16의 뉴클레오티드 서열의 프라이머 세트; 서열번호 17 및 18의 뉴클레오티드 서열의 프라이머 세트; 서열번호 19 및 20의 뉴클레오티드 서열의 프라이머 세트; 서열번호 21 및 22의 뉴클레오티드 서열의 프라이머 세트; 서열번호 23 및 24의 뉴클레오티드 서열의 프라이머 세트; 서열번호 25 및 26의 뉴클레오티드 서열의 프라이머 세트를 각각 프라이머; 및 서열번호 27 및 28의 뉴클레오티드 서열의 프라이머 세트를 각각 프라이머로 하여 PCR을 수행하여, 서열번호 8 내지 14 유전자를 각각 증폭하여 수득하였다.
번호 | 정방향 프라이머 서열 | 역방향 프라이머 서열 | |
1 | T2 | 서열번호 15 | 서열번호 16 |
2 | T4 | 서열번호 17 | 서열번호 18 |
3 | T5 | 서열번호 19 | 서열번호 20 |
4 | T6 | 서열번호 21 | 서열번호 22 |
5 | T8 | 서열번호 23 | 서열번호 24 |
6 | T9 | 서열번호 25 | 서열번호 26 |
7 | T10 | 서열번호 27 | 서열번호 28 |
증폭된 서열번호 8 유전자 (T2), 서열번호 9 유전자 (T4), 서열번호 10 유전자 (T5), 서열번호 11 유전자 (T6), 서열번호 12 유전자 (T8), 서열번호 13 유전자 (T9), 및 서열번호 14 유전자 (T10)를, 제한효소 NcoI 및 XhoI를 사용하여 절단된 pET28a (Novagen, Cat. No.69864-3)와 InFusion Cloning Kit (Clontech Laboratories, Inc.)를 통해 연결하여, T2, T4, T5, T6, T8, T9, 및 T10 유전자 발현용 벡터 pET28a-PE를 제작하였다. 이들을 각각 pT4, pT5, pT6, pT8, pT9, 및 pT10로 명명하였다.
도 1은 외래 유전자가 도입된 pET28a-PE (pT2, pT4, pT5, pT6, pT8, pT9, 및 pT10)의 벡터 지도를 나타낸다. 도 1에서, exogenous gene (T2, T4, T5, T6, T8, T9, T10)은 서열번호 8 내지 14의 외래 유전자일 수 있다.
다음으로, 제작된 pT2, pT4, pT5, pT6, pT8, pT9, 및 pT10 벡터를 각각 대장균 BL21 균주에 열충격 (heat shock) 방법 (Sambrook, J & Russell, D.W., New York: Cold Spring Harbor Laboratory Press, 2001)에 의하여 도입하고, 카나마이신 (Kanamycin, 50㎍/mL)이 포함된 LB 평판 배지 중에서 배양하여, 카나마이신 내성을 보이는 균주를 선별하였다. 최종적으로 선별된 균주를 각각 재조합 대장균 BL21/pT2, BL21/pT4, BL21/pT5, BL21/pT6, BL21/pT8, BL21/pT9, BL21/pT10로 명명하였다.
(2) 서열번호 1 내지 7의 폴리펩티드를 발현하는 재조합 대장균의 고분자 물질 분해 효과 평가
(1)절에서 제작된 서열번호 8 내지 14가 도입된 대장균 BL21/pT2, BL21/pT4, BL21/pT5, BL21/pT6, BL21/pT8, BL21/pT9, BL21/pT10 균주가 고분자 물질을 분해하는데 미치는 효과를 확인하였다.
구체적으로, 각각의 대장균 BL21/pT2, BL21/pT4, BL21/pT5, BL21/pT6, BL21/pT8, BL21/pT9, BL21/pT10 균주를 TB 배지에서 30℃에서 230rpm으로 교반하면서 배양하여, OD600이 0.5 정도에서 IPTG 0.2mM를 첨가한 후, 20℃에서 230rpm으로 교반하면서 밤새 (overnight) 배양하였다.
세포를 배양액으로부터 원심분리하여 회수 (harvest)하였다. BugBuster 단백질 추출 시약 (Novagen)을 사용하여 대장균 세포벽을 파쇄하고, 조추출물 (crude extract)을 수득하였다. 조추출물 내 단백질의 아미노산 서열 및 로커스 태그(locus tag)를 하기 표 2에 나타내었다. 로커스 태그는 GenomeNet, NCBI 등을 통해서 확인할 수 있다. 조추출물 내 총 단백질의 농도를 Bradford 방법으로 정량하였다. 조추추출물 내 총 단백질 농도 및 폴리에틸렌 분해시에 사용한 단백질의 양을 하기 표 3에 나타내었다.
번호 | 로커스 태그(locus_tag) 번호 | 아미노산 서열 | |
1 | T2 | QF06_08435 | 서열번호 1 |
2 | T4 | QF06_12865 | 서열번호 2 |
3 | T5 | QF06_04115 | 서열번호 3 |
4 | T6 | QF06_11550 | 서열번호 4 |
5 | T8 | QF06_15385 | 서열번호 5 |
6 | T9 | QF06_16355 | 서열번호 6 |
7 | T10 | QF06_01040 | 서열번호 7 |
번호 | 총 단백질 농도(mg/mL) | 폴리에틸렌 분해시에 사용한 단백질 양 (mg) | |
1 | T2 | 31.4 | 62.8 |
2 | T4 | 35.5 | 106.7 |
3 | T5 | 13.9 | 27.8 |
4 | T6 | 38.9 | 77.9 |
5 | T8 | 24.4 | 73.2 |
6 | T9 | 11.0 | 22.0 |
7 | T10 | 10.0 | 20.0 |
(3) 고분자 물질의 인장강도 변화 측정
중량평균분자량(Mw)이 155,000, 가로 x 세로의 규격이 1cm x 5cm, 질량이 약 30mg인, 폴리에틸렌 필름 3 조각 (pieces)과, (2) 절에서 수득한 T2의 단백질 조추출물 62.8mg을 함유하는 pH 7.4의 인산 완충 식염수 (Phosphate buffer saline : PBS) 10mL를 15mL의 튜브에 넣고, 30℃에서 120 rpm으로 교반하면서 7일 동안 반응시켰다. 이러한 과정을 3회 반복 (Triplicate)하였다. 대조군은 pET28a 공벡터를 대장균 BL21 균주에 열충격 방법으로 도입하여 배양된 균주로부터 수득한 조추출물을 사용하였다. T4, T5, T8, T9, T10 각각에 대하여, 표 2에 기재된 단백질 양을 사용한 것을 제외하고, 동일한 과정으로 반응시켰다. 반응이 끝난 폴리에틸렌 필름 조각을 2% SDS (sodium dodecyl sulfate) 용액으로 4시간 동안 세척한 후, 탈이온수 (deionized water)로 세정하고 밤새 상온에서 건조시켰다.
건조시킨 끝난 폴리에틸렌 필름 조각을 Texture analyzer (TA.XT plus, Stable Micro Systems) 기기를 이용하여 5 mm/min으로 상기 시료를 인장하여 파단시의 응력인 인장강도를 산출하였다. 표 4은 상기 T2, T4, T5, T8, T9, T10 및 대조군의 재조합 단백질과 폴리에틸렌을 반응시킨 후, 상기 폴리에틸렌의 인장강도의 변화를 나타낸다.
번호 | 샘플 | 인장강도(MPa) | 인장강도(%) |
1 | 대조군 | 11.52 | 100 |
2 | T2 | 8.36 | 72.57 |
3 | T4 | 10.19 | 88.42 |
4 | T5 | 10.10 | 87.65 |
5 | T8 | 9.54 | 82.79 |
6 | T9 | 9.23 | 80.12 |
7 | T10 | 9.04 | 78.50 |
표 4에 나타낸 바와 같이, T2, T4, T5, T8, T9 또는 T10 재조합 단백질을 포함하는 조추출물은 대조군에 비하여 폴리에틸렌의 인장강도를 현저하게 감소시켰다.
(3) 적외선 분광 변화 측정
폴리에틸렌의 C-H 결합 또는 C-C 결합이 C=O 결합으로 전환되었는지 여부는 푸리에 변환 적외선 분광 (Fourier Transform Infrared Spectrometry: FTIR) (Varian 670 IR, Agilent, CA, USA) 분석으로 확인하였다. 분광 분석은, Pike사(PA, USA)의 MiracleTM 악세서리를 사용하여 감쇠 전반사법 (attenuated total reflection mode: ATR)으로 측정하였다. 빛의 전반사를 위해서 굴절률 4의 값을 갖는 저마늄 크리스탈을 사용하였으며, 액화 질소를 넣은 Mercury cadmium telluride (MCT, HgCdTe) 검출기를 이용하여 상온에서 폴리에틸렌의 빛 흡수 특성을 측정하였다. 4000 내지 650 cm-1의 측정 범위 대에서 64번 스캔하여 적외선에 대한 유기 물질 (폴리에틸렌)의 흡수 스펙트럼을 얻었다.
도 3은 T4, T6, T8 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다. 도 4는 T9 및 대조군 단백질을 포함하는 조추출물이 폴리에틸렌의 C=O 결합을 증가시킨 결과를 확인한 그래프이다. T2, T4, T5, T8, T9 또는 T10 단백질을 포함하는 조추출물은 대조군에 비하여 스펙트럼 상의 1725cm-1에서 피크를 선명하게 나타내는 것으로부터, C=O 결합이 증가한 것을 알 수 있다.
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Val Gln Gln Lys Gln Met Asn Lys Ile Asp Thr Ser Leu Val Gln Ile
260 265 270
Glu Lys Glu Met Val Lys Leu Asp Glu Ile Ala Gln Gln Ile Glu Lys
275 280 285
Ile Phe Gly Ile Val Thr Gly Ile Ala Glu Gln Thr Asn Leu Leu Ser
290 295 300
Leu Asn Ala Ser Ile Glu Ser Ala Arg Ala Gly Glu His Gly Lys Gly
305 310 315 320
Phe Ala Val Val Ala Asn Glu Val Arg Lys Leu Ser Glu Asp Thr Lys
325 330 335
Lys Thr Val Ser Thr Val Ser Glu Leu Val Asn Asn Thr Asn Thr Gln
340 345 350
Ile Asn Ile Val Ser Lys His Ile Lys Asp Val Asn Glu Leu Val Ser
355 360 365
Glu Ser Lys Glu Lys Met Thr Gln Ile Asn Arg Leu Phe Asp Glu Ile
370 375 380
Val His Ser Met Lys Ile Ser Lys Glu Gln Ser Gly Lys Ile Asp Val
385 390 395 400
Asp Leu Gln Ala Phe Leu Gly Gly Leu Gln Glu Val Ser Arg Ala Val
405 410 415
Ser His Val Ala Ala Ser Val Asp Ser Leu Val Ile Leu Thr Glu Glu
420 425 430
<210> 4
<211> 347
<212> PRT
<213> Bacillus sp. YP1
<400> 4
Met Asn Glu Phe Met Lys Lys Phe Ser Leu Thr Lys Pro Ile Ile Gln
1 5 10 15
Ala Pro Met Ala Gly Gly Ile Thr Thr Pro Arg Leu Ala Ser Ala Val
20 25 30
Ser Asn Gln Gly Ala Leu Gly Ser Leu Ala Ser Gly Tyr Leu Thr Pro
35 40 45
Asp Leu Leu Glu Gln Gln Ile Lys Glu Met Phe Glu Leu Thr Asp Ala
50 55 60
Pro Phe Gln Ile Asn Val Phe Val Pro Leu Gly Leu Glu Met Pro Pro
65 70 75 80
Glu Asp Gln Ile Lys Lys Trp Lys Glu Asn Ile Pro Leu Ala Asn Gln
85 90 95
Val Asn Gln Phe Thr Ser Val Gln Glu Glu Trp Asp Asp Phe Tyr Gln
100 105 110
Lys Ile Asp Leu Ile Leu Lys Tyr Lys Val Lys Ala Cys Ser Phe Thr
115 120 125
Phe Asp Leu Pro Pro Glu Asp Ala Val Lys Glu Leu Lys Thr Ala Gly
130 135 140
Cys Cys Leu Ile Gly Thr Ala Ser Thr Val Glu Glu Ala Leu Leu Met
145 150 155 160
Glu Glu Arg Gly Met Asp Ile Val Val Leu Gln Gly Ser Glu Ala Gly
165 170 175
Gly His Arg Gly Ala Phe Leu Pro Ser Lys Gly Glu Ser Ala Val Gly
180 185 190
Leu Met Ala Leu Ile Pro Gln Ala Ala Asp Ala Leu Ser Val Pro Val
195 200 205
Ile Ala Ala Gly Gly Met Ile Asp His Arg Gly Val Lys Ala Ala Leu
210 215 220
Thr Leu Gly Ala Gln Gly Val Gln Ile Gly Ser Ala Phe Leu Ile Cys
225 230 235 240
His Glu Ser Asn Ala His Pro Val His Lys Gln Lys Ile Leu Glu Ala
245 250 255
Asn Glu Ala Asp Thr Lys Leu Thr Thr Leu Phe Ser Gly Lys Glu Ala
260 265 270
Arg Gly Ile Val Asn Lys Trp Met Glu Glu Lys Glu Gln Phe Glu Thr
275 280 285
Gln Thr Leu Pro Tyr Pro Tyr Gln Asn Thr Leu Thr Lys Ala Met Arg
290 295 300
Gln Lys Ala Ser Leu Gln Asn Asn His Asp Gln Met Ser Leu Trp Ala
305 310 315 320
Gly Gln Gly Ile Arg Ser Leu Thr Glu Glu Ile Ser Val Lys Gln Leu
325 330 335
Leu Asn Gln Leu Cys Gln Glu Asp Ile Lys Ile
340 345
<210> 5
<211> 447
<212> PRT
<213> Bacillus sp. YP1
<400> 5
Met Lys Asn Asp Asn Gln Thr Leu Lys Arg Thr Met Thr Ser Arg His
1 5 10 15
Ile Met Met Met Ala Leu Gly Gly Ala Ile Gly Ala Gly Leu Phe Lys
20 25 30
Gly Ser Ser Ser Ala Ile Asp Val Ala Gly Pro Ser Val Ile Ile Ala
35 40 45
Tyr Leu Leu Gly Gly Ile Ile Leu Leu Phe Ile Met Gln Gly Leu Ala
50 55 60
Glu Met Ala Val Arg Asn Arg Asn Ala Arg Thr Phe Arg Asp Leu Val
65 70 75 80
Gln Gln Val Leu Gly Asn Tyr Ala Ala Tyr Phe Leu Asp Trp Ile Tyr
85 90 95
Trp Lys Met Trp Val Leu Asn Ile Ala Ala Glu Ala Val Val Ala Ala
100 105 110
Ile Phe Ile Gln Tyr Trp Leu Pro Gly Cys Pro Ile Trp Val Leu Ala
115 120 125
Leu Gly Ile Ser Leu Ile Val Thr Ile Val Asn Leu Leu Ser Val Lys
130 135 140
Ile Phe Ala Glu Thr Glu Tyr Trp Leu Ala Met Ile Lys Ile Thr Val
145 150 155 160
Ile Ile Ile Phe Ile Ile Leu Gly Leu Leu Leu Leu Phe Val Ser Phe
165 170 175
Gly Asp His Thr Ala Ser Arg Phe Ser Asn Leu Thr Asp His Gly Gly
180 185 190
Phe Phe Pro His Gly Gly Thr Gly Leu Ile Thr Ala Met Leu Val Val
195 200 205
Ile Tyr Ser Tyr Gly Gly Thr Glu Ile Ile Gly Val Thr Leu Ala Glu
210 215 220
Thr Lys Asn Pro Glu Lys Val Val Pro Lys Ala Val Arg Ser Thr Leu
225 230 235 240
Thr Arg Ile Val Ala Phe Tyr Leu Leu Pro Phe Phe Ile Ile Val Ser
245 250 255
Leu Ile Pro Trp Asn Gln Val Asn Ser Val Pro Glu Ser Pro Phe Val
260 265 270
Met Val Phe Lys Met Val Gly Ile Pro Gly Ala Asp His Ile Met Asn
275 280 285
Ala Val Ile Leu Leu Ala Ile Ile Ser Ser Met Asn Ser Gly Leu Tyr
290 295 300
Gly Ser Ser Arg Ile Leu Tyr Thr Gln Ala Ser Asp Gly Arg Leu Pro
305 310 315 320
Lys Val Phe Ser Lys Leu Ser Ser Lys Asn Val Pro Met Phe Ala Ile
325 330 335
Leu Met Cys Thr Ser Ser Leu Tyr Ile Gly Val Leu Ile Ser Leu Phe
340 345 350
Ala Gly Ser Gln Thr Phe Asn Tyr Leu Met Gly Ser Leu Gly Tyr Thr
355 360 365
Val Leu Phe Ile Trp Leu Ile Ile Gly Phe Ala His Leu Lys Ser Arg
370 375 380
Lys Gln Gln Thr Glu Thr Pro Ala Tyr Tyr Val Lys Trp Phe Pro Tyr
385 390 395 400
Thr Thr Trp Phe Ala Ile Val Ala Leu Leu Ala Ile Leu Ile Gly Val
405 410 415
Ile Met Thr Thr Ser Ile Val Ile Thr Gly Ile Thr Ala Ala Ile Tyr
420 425 430
Leu Leu Ile Thr Val Ala Tyr Leu Val Lys Gly Arg Lys His Gln
435 440 445
<210> 6
<211> 380
<212> PRT
<213> Bacillus sp. YP1
<400> 6
Met Lys Lys Glu Leu Ser Phe His Glu Lys Leu Leu Lys Leu Thr Lys
1 5 10 15
Gln Gln Lys Lys Lys Thr Asn Lys His Val Phe Ile Ala Ile Pro Ile
20 25 30
Val Phe Val Leu Met Phe Ala Phe Met Trp Ala Gly Lys Ala Glu Thr
35 40 45
Pro Lys Val Lys Thr Tyr Ser Asp Asp Val Leu Ser Ala Ser Phe Val
50 55 60
Gly Asp Ile Met Met Gly Arg Tyr Val Glu Lys Val Thr Glu Gln Lys
65 70 75 80
Gly Ala Asp Ser Ile Phe Gln Tyr Val Glu Pro Ile Phe Arg Ala Ser
85 90 95
Asp Tyr Val Ala Gly Asn Phe Glu Asn Pro Val Thr Tyr Gln Lys Asn
100 105 110
Tyr Lys Gln Ala Asp Lys Lys Ile His Leu Gln Thr Asn Lys Glu Ser
115 120 125
Val Lys Val Leu Lys Asp Met Asn Phe Thr Val Leu Asn Ser Ala Asn
130 135 140
Asn His Ala Met Asp Tyr Gly Val Gln Gly Met Lys Asp Thr Leu Gly
145 150 155 160
Glu Phe Ala Lys Gln Asn Leu Asp Ile Val Gly Ala Gly Tyr Ser Leu
165 170 175
Ser Asp Ala Lys Lys Lys Ile Ser Tyr Gln Lys Val Asn Gly Val Thr
180 185 190
Ile Ala Thr Leu Gly Phe Thr Asp Val Ser Gly Lys Gly Phe Ala Ala
195 200 205
Lys Lys Asn Thr Pro Gly Val Leu Pro Ala Asp Pro Glu Ile Phe Ile
210 215 220
Pro Met Ile Ser Glu Ala Lys Lys His Ala Asp Ile Val Val Val Gln
225 230 235 240
Ser His Trp Gly Gln Glu Tyr Asp Asn Asp Pro Asn Asp Arg Gln Arg
245 250 255
Gln Leu Ala Arg Ala Met Ser Asp Ala Gly Ala Asp Ile Ile Val Gly
260 265 270
His His Pro His Val Leu Glu Pro Ile Glu Val Tyr Asn Gly Thr Val
275 280 285
Ile Phe Tyr Ser Leu Gly Asn Phe Val Phe Asp Gln Gly Trp Thr Arg
290 295 300
Thr Arg Asp Ser Ala Leu Val Gln Tyr His Leu Lys Lys Asn Gly Thr
305 310 315 320
Gly Arg Phe Glu Val Thr Pro Ile Asp Ile His Glu Ala Thr Pro Ala
325 330 335
Pro Val Lys Lys Asp Ser Leu Lys Gln Lys Thr Ile Ile Arg Glu Leu
340 345 350
Thr Lys Asp Ser Asn Phe Ala Trp Lys Val Glu Asp Gly Lys Leu Thr
355 360 365
Phe Asp Ile Asp His Ser Asp Lys Leu Lys Ser Lys
370 375 380
<210> 7
<211> 95
<212> PRT
<213> Bacillus sp. YP1
<400> 7
Met Val Arg Glu Ala Ala Met Leu His Ile Lys Glu Gly Leu Glu Gln
1 5 10 15
Glu Phe Glu Asp Ala Phe Arg Gln Ala Ala Pro Ile Ile Ser Gly Met
20 25 30
Lys Gly Tyr Ile Thr His Ser Leu Ser Lys Cys Met Glu Glu Thr His
35 40 45
Lys Tyr Leu Leu Leu Val Glu Trp Glu Thr Leu Glu Asn His Thr Glu
50 55 60
Gly Phe Arg Gly Ser Ser Glu Tyr Gln Glu Trp Lys Ala Leu Leu His
65 70 75 80
Arg Phe Tyr Thr Pro Phe Pro Thr Val Glu His Phe Gln Asp Val
85 90 95
<210> 8
<211> 1452
<212> DNA
<213> Bacillus sp. YP1
<400> 8
atgggtatca tcaacggtaa agagttcatc gaccgcctga acaagctgga gaatgagatc 60
tggtatgacg gcgagaagat caagggtaac atcagcgagc accctgcatt caaaggtatc 120
attaagacca agtccagcct gtacgagctg caaaccaaag acgagctgat ccacgaaatg 180
acgtactgcc tgcctggtga tcataatcgt atcggtctgt cctacctgca gccaaaaacg 240
aaaaacgacc tgaagaaacg ccgtactatg atcgagcact gggctcgtca tactcacggt 300
atgatgggtc gttctccaga ttacatgaac actgtcatga tgagcttcgc ttccagcgca 360
gaactgctga aagataaaga aaactgcttc ccggaacaca tcctggacat gtacgaacag 420
gctgctaaac acgacctgtc cttcactcac actttcatca ccccacaggt aaaccgtagc 480
cagtcttact tcggcctgtc tgaaaaaccg atctccgcta aagtaatcga tcgcactgaa 540
aagggtctga tgatccatgg cgcacgtctg ctggcaactc aaggtggtct gactgatgaa 600
attctggttt tcagcgcacc gaaattcttt tttgaaaccg acgaagctta cgctttctcc 660
atcccgtcta acactaaagg tgtgaaattc attacccgcg agagcttcgt gctgagcgac 720
tcttctttca accacccgct gtcttctcgt tacgaagaaa tggactctat cgtcgtcttc 780
gaccacgtac tggtaccgtg gaaccgtgtt ttcttctacg acaacgttga agcggcgaaa 840
gacttcatga ccaaaagctc cttccacgca ttcaccttcc atcaggttgt tatccgtcag 900
atgatcaaaa tcgaattcct gctgggcgtg gcccagctgc tggttgatac catcaacgtt 960
tctgaatacc agcacatcca ggaaaaactg tccgaaatta tcgtgggcct ggaaaccatc 1020
aaagccctga ttgacaaatc cgaaaacgac gcgcagctgg atgaattcgg ctacatgcgc 1080
ccgtgtctga ttccgctgca agttatctct acgatcattc cgaaactgta cccgcgtttt 1140
acggaaatta ttcagctgat tggcgcgagc ggcatggtga ccctgccgac cgaaaatgcg 1200
tttgactctg aaatccgtga agatctggat caatatctgc aggccaccaa caccaacgcg 1260
gaagaacgtg tgaaaatttt tcgtctggcc tgggatctga ccatgtcttc ttttggcacg 1320
cgtcagaccc attatgaacg ctattttttt ggcgatccga ttcgcatttc ctcccgcctg 1380
tatacctcct atccgaaaca ggaacagctg aacatgatta aaacctttct gcacgcggat 1440
gcggaacagt aa 1452
<210> 9
<211> 1329
<212> DNA
<213> Bacillus sp. YP1
<400> 9
atgacccgta gcgatttcat ccagttcggt gcaatgatcc acggtgtcgg tggcactact 60
gacggttggc gtcatcctga tgttgatcct tctgcatcta cgaacatcga attctacatg 120
aagaaggccc agaccgctga aaagggtctg ttctccttca tcttcatcgc ggacggcctg 180
ttcatcagcg agaagtccat ccctcacttc ctgaaccgtt tcgagccaat caccatcctg 240
agcgcactgg catctgtcac taagaacatt ggtctggtcg gtacgttctc caccagcttc 300
accgagccat tcaccatctc ccgtcagctg atgtctctgg atcacatctc cggtggtcgt 360
gcaggttgga atctggttac gagcccacaa gaaggtgcag cacgtaatca cagcaaaagc 420
aacctgccgg aacacactga gcgttacgag atcgcacagg aacacctgga tgtggttcgt 480
ggtctgtgga actcttggga acacgatgct ttcattcaca acaaaaaaac cggtcagttc 540
ttcgacccgg cgaaactgca tcgcctgaac cacaaaggca aatacttcca ggtggagggt 600
ccgctgaaca tcggccgctc taaacagggc gaaccggtgg tattccaggc tggttcttct 660
gaaaccggcc gtcaattcgc tgctaaaaac gcagacgcta tcttcaccca ctccaactcc 720
ctggaagaaa ccaaagcgtt ctacgcggac gtgaaacgcc gcgctgctga taaaggtcgt 780
gacccgtctt ctgtacgtat ttttccgggt atcagcccga tcgtagctga cacggaagag 840
gaggctgaaa aaaaataccg cgaatttgcc gaactgatcc cgatcgaaaa cgcggtgacc 900
tacctggccc gcttctttga cgactacgac ctgtccgtgt atccgctgga cgaaccgttc 960
ccggacatcg gtgatgttgg caaaaatgcg tttcagagca ccaccgaccg tatcaaacgc 1020
gaagcgaaag cccgtaacct gactctgcgt gaagttgcgc aagaaatggc gtttccgcgt 1080
ccgctgttta tcggcactcc ggaacgtgtt gcctctctga ttgaaacttg gtttaacgcg 1140
gaagccgcgg atggctttat tattggctcc gacattccgg gcaccctgga tgcgtttgta 1200
gaaaaagtta ttccgattct gcaggaacgt ggcctgtatc gtcaggatta tcgcggcggc 1260
actctgcgcg aaaacctggg cctgggcatt ccgcagcatc agtccgttct gcattctagc 1320
catcactaa 1329
<210> 10
<211> 1299
<212> DNA
<213> Bacillus sp. YP1
<400> 10
atgctgttta agaaggaccg taagcaagag actgcttact ttagcgacag caacggtcag 60
caaaagaacc gtatccagct gactaacaaa cacgcagacg taaaaaaaca gctgaaaatg 120
gtgcgcctgg gtgatgctga gctgtacgta ctggagcaac tgcagccact gatccaggaa 180
aatatcgtaa acatcgtcga cgcattctac aaaaacctgg accacgagtc ttctctgatg 240
gacatcatca acgaccattc cagcgtcgat cgtctgaaac agacgctgaa acgtcatatc 300
caggagatgt tcgcaggtgt aatcgatgat gagttcattg agaaacgcaa ccgtattgcg 360
tctatccacc tgcgtatcgg tctgctgccg aaatggtata tgggtgcatt tcaagagctg 420
ctgctgtcta tgattgacat ctacgaggca tctatcacta accagcagga actgctgaaa 480
gcaatcaaag cgactactaa aatcctgaac ctggaacagc agctggtcct ggaagctttc 540
cagtctgaat ataaccagac ccgtgacgaa caggaagaga agaagaacct gctgcaccag 600
aaaattcagg aaacctccgg ttctatcgct aacctgttct ctgaaacctc tcgttctgtg 660
caagaactgg tcgacaagtc tgaaggtatc tctcaggctt ctaaagctgg tactgtgacc 720
tcttccactg tggaagaaaa gtccatcggt ggcaaaaaag agctggaagt tcagcagaaa 780
cagatgaaca aaatcgacac cagcctggtt cagattgaaa aagaaatggt gaaactggac 840
gaaatcgccc agcagatcga aaaaatcttc ggcatcgtga ccggcatcgc ggaacagacc 900
aatctgctgt ccctgaacgc ttccatcgaa agcgcccgtg ccggtgaaca tggcaaaggc 960
ttcgcggttg ttgcgaatga agttcgcaaa ctgagcgaag atacgaaaaa aaccgtttcc 1020
accgtttctg aactggttaa caacaccaac acgcaaatta atatcgtgtc caaacacatt 1080
aaagatgtga acgaactggt gagcgaatcc aaagaaaaaa tgacccagat taaccgcctg 1140
ttcgatgaaa ttgttcacag catgaaaatt agcaaagaac agagcggcaa aattgatgtt 1200
gatctgcagg cgttcctggg cggcctgcag gaagtaagcc gcgccgtttc ccacgttgcg 1260
gcgtccgttg attccctggt aattctgacc gaagaataa 1299
<210> 11
<211> 1044
<212> DNA
<213> Bacillus sp. YP1
<400> 11
atgaacgagt tcatgaagaa gttcagcctg accaagccga tcatccaggc tccaatggct 60
ggcggtatca ccactcctcg tctggcatct gcagtttcta accagggtgc tctgggttct 120
ctggcttctg gttacctgac tccagatctg ctggaacaac agatcaagga gatgttcgag 180
ctgaccgatg caccatttca gattaacgtg ttcgtgccgc tgggtctgga gatgccgcct 240
gaagatcaaa tcaagaaatg gaaagagaac atcccgctgg ctaaccaagt caaccagttc 300
acctccgtcc aggaagaatg ggacgacttc taccagaaaa tcgacctgat cctgaaatac 360
aaagtgaaag cttgcagctt caccttcgac ctgccgccgg aagacgccgt gaaagaactg 420
aaaaccgctg gttgctgtct gatcggcacc gctagcaccg tggaggaagc cctgctgatg 480
gaggaacgcg gtatggacat cgttgtactg cagggttctg aagcaggtgg tcaccgtggt 540
gcattcctgc cgtctaaagg tgaatctgct gttggtctga tggcactgat cccgcaagca 600
gccgatgcgc tgagcgttcc ggtaattgca gcgggcggca tgattgatca tcgtggcgtt 660
aaagcggcgc tgactctggg cgcgcagggc gttcagattg gctccgcctt tctgatttgt 720
catgaatcca atgcgcaccc ggtacacaaa cagaaaattc tggaagcgaa tgaagcggat 780
actaaactga ctactctgtt tagcggcaaa gaagcccgtg gcattgtaaa caaatggatg 840
gaagaaaaag aacagtttga aacccagacc ctgccgtatc cgtatcagaa cacgctgacg 900
aaagcgatgc gccagaaagc gagcctgcag aacaaccacg accagatgtc cctgtgggcg 960
ggccagggca tccgttccct gacggaagaa atctccgtta aacagctgct gaaccagctg 1020
tgccaagaag acatcaaaat ctaa 1044
<210> 12
<211> 1344
<212> DNA
<213> Bacillus sp. YP1
<400> 12
atgaaaaacg acaaccagac gctgaagcgt acgatgacgt cccgtcacat tatgatgatg 60
gcgctgggtg gtgcaatcgg tgcaggtctg ttcaaaggtt cttcttccgc aatcgatgtg 120
gcaggtccat ctgtaatcat cgcatacctg ctgggcggta tcattctgct gttcatcatg 180
cagggtctgg cagaaatggc tgtccgtaac cgtaacgcac gtactttccg tgatctggtg 240
cagcaggtgc tgggtaacta tgctgcttac tttctggatt ggatctactg gaagatgtgg 300
gtcctgaaca tcgctgcgga agctgttgta gctgctatct tcattcagta ctggctgccg 360
ggttgtccta tttgggtact ggcactgggt attagcctga tcgttactat cgtgaacctg 420
ctgtctgtca agatcttcgc ggagactgag tattggctgg caatgatcaa aatcaccgtc 480
atcattatct ttattatcct gggcctgctg ctgctgtttg tgagcttcgg cgaccacacc 540
gcgtcccgtt tctccaacct gactgaccac ggtggtttct tccctcacgg tggcaccggt 600
ctgattactg ccatgctggt agttatttac tcttacggcg gtactgaaat cattggcgtg 660
actctggctg aaactaaaaa cccggaaaaa gtcgttccga aagcggtgcg tagcactctg 720
acccgtatcg tggctttcta tctgctgccg ttcttcatca tcgtgtccct gatcccatgg 780
aatcaggtga acagcgttcc ggaatctccg tttgtaatgg ttttcaaaat ggtaggcatc 840
ccgggcgctg accatatcat gaacgccgtt atcctgctgg ctatcatcag cagcatgaac 900
tccggtctgt acggtagcag ccgcattctg tacacccaag cctctgatgg tcgcctgccg 960
aaagtttttt ctaaactgtc ttccaaaaat gtaccgatgt tcgccatcct gatgtgcacc 1020
tccagcctgt atatcggcgt actgatctcc ctgttcgcgg gctctcaaac cttcaattac 1080
ctgatgggct ccctgggcta taccgttctg ttcatttggc tgattatcgg cttcgcgcat 1140
ctgaaatctc gcaagcagca gaccgaaacc ccggcctatt atgttaaatg gtttccgtac 1200
accacctggt ttgcgatcgt tgcgctgctg gccattctga ttggcgttat catgaccacc 1260
tctatcgtta tcacgggcat taccgcggcg atttacctgc tgattaccgt tgcgtacctg 1320
gttaaaggcc gcaaacacca gtaa 1344
<210> 13
<211> 1143
<212> DNA
<213> Bacillus sp. YP1
<400> 13
atgaagaagg agctgagctt ccacgaaaag ctgctgaagc tgactaagca gcaaaaaaag 60
aagacgaaca agcacgtttt catcgcaatc ccgatcgttt tcgtcctgat gttcgctttc 120
atgtgggcag gtaaagcaga gactcctaaa gtaaaaacct acagcgacga cgtactgagc 180
gcttcttttg tgggtgatat catgatgggt cgttacgtcg agaaagtgac tgagcagaaa 240
ggtgcagata gcatcttcca gtacgttgaa ccaatcttcc gtgcttccga ttacgtggct 300
ggtaacttcg aaaacccggt aacttaccag aaaaactaca aacaggcaga caaaaaaatc 360
cacctgcaga ccaacaaaga atccgttaaa gtgctgaaag acatgaactt caccgtgctg 420
aacagcgcga acaaccacgc gatggactac ggcgtgcagg gtatgaaaga taccctgggt 480
gaattcgcaa aacagaacct ggacatcgtg ggtgcaggtt actctctgtc tgacgcgaaa 540
aagaaaatct cctatcagaa agttaacggc gtgacgatcg cgactctggg tttcactgac 600
gtatctggta aaggtttcgc tgctaaaaaa aacaccccgg gtgtgctgcc agctgatcct 660
gaaattttca tcccgatgat ctctgaagcg aaaaaacatg ccgacatcgt tgtcgtccag 720
tcccactggg gccaagaata tgacaacgac ccgaatgacc gtcagcgtca actggctcgt 780
gccatgtctg atgcgggcgc ggatattatt gttggccatc atccgcatgt tctggaaccg 840
attgaagttt ataatggcac tgttattttt tattctctgg gcaacttcgt atttgaccag 900
ggctggaccc gtacccgcga ttctgcgctg gttcagtatc acctgaaaaa aaatggcacc 960
ggccgctttg aagtaacccc gattgatatt cacgaagcga ccccggcccc ggttaaaaaa 1020
gatagcctga aacagaaaac cattatccgc gaactgacca aagattccaa ctttgcctgg 1080
aaagttgaag atggcaaact gacgtttgat atcgaccact ccgacaaact gaaatccaaa 1140
taa 1143
<210> 14
<211> 288
<212> DNA
<213> Bacillus sp. YP1
<400> 14
atggtcagag aggcagcaat gcttcacatt aaagaaggcc ttgaacaaga gtttgaggat 60
gcattccggc aagcggcgcc gattatttcc ggcatgaagg gctatatcac tcactcgtta 120
tcaaaatgca tggaggaaac gcataaatat ctgcttcttg tggagtggga aacacttgag 180
aaccatacgg aaggctttcg cggttcttca gagtatcaag agtggaaagc cttgcttcat 240
cgattctata ccccgttccc gacggtcgag cattttcagg atgtgtaa 288
<210> 15
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> T2 F-primer
<400> 15
aagaaggaga tataccatgg gtatcatcaa cggtaaag 38
<210> 16
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> T2 R-primer
<400> 16
gtggtggtgg tggtgctcga ttactgttcc gcatccgcgt 40
<210> 17
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T4 F-primer
<400> 17
aagaaggaga tataccatga cccgtagcga tttcatc 37
<210> 18
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> T4 R-primer
<400> 18
gtggtggtgg tggtgctcga ttagtgatgg ctagaatgc 39
<210> 19
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T5 F-primer
<400> 19
aagaaggaga tataccatgc tgtttaagaa ggaccgt 37
<210> 20
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> T5 R-primer
<400> 20
gtggtggtgg tggtgctcga ttattcttcg gtcagaatta c 41
<210> 21
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T6 F-primer
<400> 21
aagaaggaga tataccatga acgagttcat gaagaag 37
<210> 22
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> T6 R-primer
<400> 22
gtggtggtgg tggtgctcga ttagattttg atgtcttctt g 41
<210> 23
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T8 F-primer
<400> 23
aagaaggaga tataccatga aaaacgacaa ccagacg 37
<210> 24
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> T8 R-primer
<400> 24
gtggtggtgg tggtgctcga ttactggtgt ttgcggcct 39
<210> 25
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T9 F-primer
<400> 25
aagaaggaga tataccatga agaaggagct gagcttc 37
<210> 26
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> T9 R-primer
<400> 26
gtggtggtgg tggtgctcga ttatttggat ttcagtttg 39
<210> 27
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> T10 F-primer
<400> 27
aagaaggaga tataccatgg tcagagaggc agcaatg 37
<210> 28
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> T10 R-primer
<400> 28
gtggtggtgg tggtgctcga ttacacatcc tgaaaatgc 39
Claims (18)
- 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질 또는 이들의 조합.
- 청구항 1에 있어서, 옥시게나제 활성을 갖는 것인 재조합 단백질 또는 이들의 조합.
- 청구항 1에 있어서, 고분자 물질에 카르보닐기를 도입하는 것을 촉매하는 활성을 갖는 것인 재조합 단백질 또는 이들의 조합.
- 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 재조합 단백질을 코딩하는 뉴클레오티드 서열을 포함하는 유전자.
- 청구항 4에 있어서, 상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 어느 하나의 뉴클레오티드 서열을 포함하는 것인 유전자.
- 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물.
- 청구항 6에 있어서, 상기 재조합 미생물은 모균주에 비하여 증가된 카피 수의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를 포함하는 것인 재조합 미생물.
- 청구항 7에 있어서, 상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 하나 이상의 뉴클레오티드 서열을 포함하는 것인 재조합 미생물.
- 청구항 6에 있어서, 상기 재조합 미생물은 에스케리키아 (Escherichia) 속, 효모 (yeast), 잔토모나스 (Xanthomonas) 속, 살모넬라 (Samonellar) 속, 슈도모나스 (Pseudomonas) 속, 코리네박테리움 (Corynebacterium) 속, 또는 바실러스 (Bacillus) 속에 속하는 것인 재조합 미생물.
- 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 미생물, 그 분해물 (lysate) 또는 그 분해물의 분획을 고분자 물질과 접촉시켜 상기 고분자 물질을 분해하는 단계를 포함하는, 고분자 물질을 분해하는 방법.
- 청구항 10에 있어서, 상기 접촉은 밀폐된 용기 중에서 수행되는 것인 방법.
- 청구항 10에 있어서, 상기 미생물은 모균주에 비하여 증가된 수준의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 포함하는 재조합 미생물인 것인 방법.
- 청구항 12에 있어서, 상기 재조합 미생물은 모균주에 비하여 증가된 카피수의 서열번호 1,2,3,4,5,6, 및 7 중 어느 하나의 아미노산 서열과 95% 이상의 서열 동일성을 갖는 아미노산 서열을 포함하는 단백질 또는 이들의 조합을 코딩하는 유전자를 포함하는 것인 방법.
- 청구항 13 있어서, 상기 유전자는 서열번호 8 내지 14의 뉴클레오티드 서열로 이루어지는 군으로부터 선택된 하나 이상의 뉴클레오티드 서열을 포함하는 것인 방법.
- 청구항 12에 있어서, 상기 재조합 미생물은 에스케리키아 (Escherichia) 속, 효모 (yeast), 잔토모나스 (Xanthomonas) 속, 살모넬라 (Samonellar) 속, 슈도모나스 (Pseudomonas) 속, 코리네박테리움 (Corynebacterium) 속, 또는 바실러스 (Bacillus) 속에 속하는 것인 방법.
- 청구항 10에 있어서, 상기 고분자 물질은 폴리알킬렌계, 폴리아크릴레이트계, 및 폴리스티렌계로부터 선택되는 1종 이상인 것인 방법.
- 청구항 16에 있어서, 상기 폴리알킬렌은 탄소수 1 내지 10의 알킬렌 반복 단위를 갖는 것인 방법.
- 청구항 16에 있어서, 상기 폴리알킬렌은 폴리에틸렌인 것인 방법.
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US15/724,979 US10465210B2 (en) | 2016-10-04 | 2017-10-04 | Recombinant protein, recombinant microorganism, and method of degrading macromolecular substance |
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