KR930701589A - 형질전환된 미생물에 의한 멜라닌의 제조방법 - Google Patents

형질전환된 미생물에 의한 멜라닌의 제조방법

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KR930701589A
KR930701589A KR1019920703432A KR920703432A KR930701589A KR 930701589 A KR930701589 A KR 930701589A KR 1019920703432 A KR1019920703432 A KR 1019920703432A KR 920703432 A KR920703432 A KR 920703432A KR 930701589 A KR930701589 A KR 930701589A
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melanin
tyrosine
tyrosinase
streptomyces
producing
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델라-시오파 구이
제이. 가거 쥬니어 스테펜
지. 스버로우 게나디
에이치. 티펜 토마스
케이. 그릴 로렌스
알. 체데켈 마일즈
에이치. 쿠마가이 몬토
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데이비드 알. 맥지
바이오소스 제네틱스 코포레이션
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Abstract

본 발명은 멜라닌의 제조방법, 활성티로시나제의 제조방법에 관한 것으로써, 본 발명에 따른 멜라닌제조방법은 멜라닌제조효소를 코딩하는 제1DNA씨퀀스와 활성제를 코딩하는 제2DNA씨퀀스인, 멜라닌를 생성하는데 유용한 둘이상의 DNA씨퀀스를 함유하도록 미생물을 형질전화시켜서 멜라닌을 생성하도록 되어있고, 상기 제1DNA씨퀀스가 티로시나제를 코딩하며, 이것은 진균, 세균, 인간, 동물 또는 식물에서 얻도록 되어있다.
또한 본 발명에 따른 활성티로시나제의 제조방법은 E. Coli세포를 티로신을 코딩하는 씨퀀스를 함유하는 DNA단편과 활성제단백질을 코딩하는 씨퀀스를 함유하는 DNA단편에 형질전환시키고, 형질전환된 E. Coli세포를 적당한 배지에서 배양하는 것으로 되어있다.

Description

형질전환된 미생물에 의한 멜라닌 제조방법
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 멜라닌 생합성에 대한 단계적 도면, 제2도는 ORF 438+티로시나제(pBGC 620.3)와 티로시나제(pBGC 619)를 포함하는 E. Coli 발현 플라스미드를 나타내는 도면 (엠피실린 내성 유전자(AMP)의 위치 T7 프로모터(T7), 그리고 리보좀 결합부(RBS1 그리고 2)가 박스내에 나타나있다. RBS1(밑줄부)으로부터 티로시나제(pBGC619)의 처름 일곱 코돈(윗쪽)이나 ORF 438(pBGC 620.3)까지의 벡터 시퀀스는 5'aaggagatatacatATGGCTAGAATTGCCATGGCC3'이다. RBS2(밑줄부)로 부터 티로시나제(pBGC 620.3)의 ATG 스타트 코돈까지의 항생물질 S의 씨퀀스는 5'-ggagcacccgcacATG-3'이다. 제3도는 ORF 438 단백질이 있거나 또는 없는 상태에서 티로시나제를 생산하는 E. coli 세포내의 SDS-파지 (A) [35S] 리팜피신이 있는 생체내에서 단백질합성의 메티오닌(Methionine) 방사능사진(1행, PBGC 619; 2행, PT-7 벡터콘트롤; 3행, PBGC 620.2; 4행, PBGC 620.3 필름은 2hr 노출.) (B) 쿠마시(Coomassie)로 착색된 젤(1행, PT7-7 벡터 콘트롤; 2행, PBGC 619; 3행, PBGC 620.2; 4행, PBGC 620.3 분자량 마커의 위치는 왼쪽, 티로시나제와 ORF 438(orf)의 위치는 오른쪽.)

Claims (42)

  1. 완화된 발효조건에서 배지의 멜라닌농도가 배지 1ℓ당 습윤중량으로 적어도 3.3g이 될때까지 멜라닌생성미생물을 배양하고, 성장배지로부터 멜라닌을 추출하는 것으로 구성되는 멜라닌제조방법에 있어서, 멜라닌 제조효소를 코딩하는 제1DNA씨퀀스와 활성제를 코딩하는 제2DNA씨퀀스인, 멜라닌을 생성하는데 유용한 둘이상의 DNA씨퀀스를 함유하도록 미생물을 형질전화시키는 것을 특징으로 하는 멜라닌제조방법.
  2. (a) 멜라닌생성효소를 코딩하는 제1DNA씨퀀스와 활성제를 코딩하는 제2DNA씨퀀스인, 멜라닌생성에 유용한 둘이상의 DNA씨퀀스를 포함하는 발현벡터를 제조하고; (b) 상기 벡터를 숙주세균에 형질전환시키고; (c) 적당한 성장배지에서 배지의 멜라닌농도가 1ℓ당 습윤중량이 적어도 3.3ℓ이 될때까지 상기 형질전환된 세균을 배양하고; (d) 상기 배지로부터 멜라닌을 추출하는 상기 단계로 구성되는 것을 특징으로 하는 멜라닌제조방법.
  3. 제1항 또는 제2항에 있어서, 상기 유전자를 코딩하는 제1DNA씨퀀스는 산화·환원효소, 티로신 가수분해 효소, 티로시나제, 라크효소, 폴리페닐옥시다아제로 구성되는 그룹으로부터 선택되는 것을 특징으로 하는 멜라닌제조방법.
  4. 제1항 또는 제2항에 있어서, 상기 제1DNA씨퀀스가 티로시나제를 코딩하는 것을 특징으로 하는 멜라닌제조방법.
  5. 제1항 내지 제4항중 어느 한항에 있어서, 상기 미생물을 상기 DNA씨퀀스의 추가적인 3'변형 또는 유전자 코딩 씨퀀스에 형질전환시키는 것을 특징으로 하는 멜라닌제조방법.
  6. 제1항 내지 제5항중 어느 한항에 있어서, 제1DNA씨퀀스의 분비가 가능한 시그날씨퀀스를 코딩하는 DNA씨퀀스로 형질전환시키는 것을 특징으로 하는 멜라닌제조방법.
  7. 제1항 내지 제6항중 어느 한항에 있어서, 상기 제1DNA씨퀀스는 진균, 세균, 인간, 동물 또는 식물로부터 얻는 것을 특징으로 하는 멜라닌제조방법.
  8. 제1항 내지 제7항중 어느 한항에 있어서, 미생물이 세균 또는 진균인 것을 특징으로하는 멜라닌제조방법.
  9. 제1항 내지 제8항중 어느 한항에 있어서, 미생물이 Streptomyces 또는 Escherichia속 세균인 것을 특징으로 하는 멜라닌제조방법.
  10. 제9항에 있어서, Streptomyces속 미생물이 Streptomyces lividans, Streptomyces scabies, Streptomyces antibiouticus, Streptomyces coelicolor, Streptomyces fradiae, Streptomyces glaucesens, Streptomyces venezullae 및 Streptomyces azureus로 구성되는 그룹으로부터 선택되는 것을 특징으로 하는 멜라닌제조방법.
  11. 제9항에 있어서, Escherichia속 미생물이 E. Coli인 것을 특징으로 하는 멜라닌제조방법.
  12. 제1항 내지 제11항중 어느 한항에 있어서, 성장배지가 하나이상의 멜라닌전구체를 포함하는 것을 특징으로 하는 멜라닌제조방법.
  13. 제12항에 있어서, 상기 전구체가 카제인 가수분해물 또는 카제인펩톤에 의해 제공되는 것을 특징으로 하는 멜라닌제조방법.
  14. 제12항에 있어서, 상기 전구체가 1-시스테인에틸에스테르, 3-니트로-L-티로신, 3-플루오로-DL-티로신, 3-메톡시-L-티로신, L-프롤린, (±)-시네프린, DL-m-티로신, 3-아이오드-L-티로신, 3, 4-디히드록시-시남산, 글리신-L-티로신, L-B-3, 4-도파메틸에스테르, DL-옥토파민, 4-히드록시인돌, 티라민, L-시스테인, L-메티오닌, 5, 6-디메톡시인돌, (-)-아테레놀, D-도파, 5-히드록시트립타민, L-티로신에틸에스테르, L-티로신 및 L-도파로 구성된 그룹에서 선택되는 것을 특징으로 하는 멜라닌제조방법.
  15. 제12항에 있어서, 상기 하나의 구체는 티로신이고, 또 하나이상의 전구체는 1-시스테인에틸에스테르, 3-니트로-L-티로신, 3-플루오로-DL-티로신, 3-메톡시-L-티로신, L-프롤린, (±)-시네프린, DL-m-티로신, 3-아이오드-L-티로신, 3, 4-디히드록시-시남산, 글리신-L-티로신, L-B-3, 4-도파메틸에스테르, DL-옥토파민, 4-히드록시인돌, 티라민, L-시스테인, L-메티오닌, 5, 6-디메톡시인돌, (-)-알테레놀, 5-히드록시트립타민, L-티로신에틸 에스테르 및 L-도파로 구성된 그룹에서 선택되는 것을 특징으로 하는 멜라닌제조방법.
  16. 제1항 내지 제15항중 어느 한항에 있어서, 미생물이 벡터 pBS1055 또는 pBS1057을 포함하는 S. lividans인 것을 특징으로 하는 멜라닌제조방법.
  17. 제16항에 있어서, 미생물이 S. lividans TK64인 것을 특징으로 하는 멜라닌제조방법.
  18. 제1항 내지 제17항중 어느 한항에 있어서, 미생물이 pGP1-2에 정박하고(harbored), pBGC620.3, pBS1012.7, pBS1018, pBS1022, pBS1024, pBS1025, pBS1026 및 pBS636중 하나이상의 벡터를 포함하는 E. Coli인 것을 특징으로 하는 멜라닌제조방법.
  19. 제18항에 있어서, 미생물이 E. Coli K-38, C600, JM101 또는 KJM109인 것을 특징으로 하는 멜라닌제조방법.
  20. 멜라닌을 생성하는데 유용한 둘이상의 DNA씨퀀스를 갖는 미생물과 성장배지를 함유하는 배양액에 있어서, 제1DNA씨퀀스는 멜라닌생성효소를 코딩하고, 제2DNA씨퀀스는 활성제를 코딩하며, 상기 배양물은 배지 1ℓ당 습윤중으로 적어도 멜라닌 3.3g을 생성할 수 있는 것을 특징으로하는 성장배지와 미생물을 함유하는 배양액.
  21. Streptomyces antibioticus ATCC 8663의 티로시나제 유전자를 함유하는 발현벡터.
  22. 제21항에 있어서, 티로시나제에 대해 양성활성작용을 갖는 단백질을 코딩하며, Streptomyces antibioticus ATCC8663으로부터 분리된 유전자씨퀀스 또한 포함하는 것을 특징으로 하는 발현벡터.
  23. 제21항 또는 제22항에 있어서, pBS1055 또는 pBS1057인 것을 특징으로하는 발현벡터.
  24. Streptomyce antiobicus ATT8663으로부터 얻은 정제된 티로시나제 단백질.
  25. Streptomyce antiobicus ATT8663으로부터 얻은 정제된 활성제단백질.
  26. Streptomyce antiobicus IMRU3720의 ORF438과 S. antibioticus ATCC8663의 ORF438유사체의 키메라의 단백질생성물인 것을 특징으로하는 정제된 활성단백질.
  27. Streptomyce antiobicus ATT8663으로부터 얻은 정제된 티로시나제 유전자.
  28. Streptomyce antiobicus ATT8663으로부터 얻은 정제된 활성제유전자.
  29. (a) E. coli세포를 티로신을 코딩하는 씨퀀스를 함유하는 DNA단편과 활성제 단백질을 코딩하는 씨퀀스를 함유하는 DNA단편에 형질전화시키고, (b) 형질전환된 E. Coli세포를 적당한 배지에서 배양하는 상기 단계로 구성된 것을 특징으로하는 활성티로시나제의 제조방법.
  30. 제29항에 있어서, 활성티로시나제를 추출하는 단계 또는 포함하는 것을 특징으로 하는 활성티로시나제의 제조방법.
  31. 제29항 또는 제30항에 있어서, 활성티로시나제의 특이활성이 티로시나제1mg당 1분에 적어도 1.50μmole의 L-도파를 산화시키는 정도인 것을 특징으로하는 활성티로시나제의 제조방법.
  32. 제29항 내지 제31항중 어느 한항에 있어서, 성장배지로부터 E. Coli세포를 제거하고, 세포를 투과할 수 있도록하는 단계 또는 포함하는 것을 특징으로하는 활성티로시나제의 제조방법.
  33. 제32항에 있어서, 투과할 수 있도록 E. Coli세포에서 티로시나제 특이활성이 세포 1㎕당 1분간 적어도 7.50μmol의 L-도파를 산화시키는 정도인 것을 특징으로하는 활성티로시나제의 제조방법.
  34. 제32항에 있어서, 투과할 수 있도록 E. Coli세포에서 티로시나제 특이활성이 세포 1㎕당 1분간 적어도 13.9μmol의 L-도파를 산화시키는 정도인 것을 특징으로하는 활성티로시나제의 제조방법.
  35. 제32항에 있어서, 투과할 수 있도록 E. Coli세포에서 티로시나제 특이활성이 세포 1㎕당 1분간 적어도 20.01μmol의 L-도파를 산화시키는 정도인 것을 특징으로하는 활성티로시나제의 제조방법.
  36. 제29항 내지 제35항중 어느 한항에 있어서, 활성단백질이 ORF438에 의해 코딩되는 것을 특징으로하는 활성티로시나제의 제조방법.
  37. 제29항 내지 제35항중 어느 한항에 있어서, Streptomyces antibioticus ATCC8663의 오프리딩프레임에 의해 코딩되는 것을 특징으로하는 활성티로시나제의 제조방법.
  38. 제29항 내지 제35항중 어느 한항에 있어서, 활성단백질이 하이브리드 코딩씨퀀스에 의해 코딩되는 것을 특징으로하는 활성티로시나제의 제조방법.
  39. (a) 티로시나제를 코딩하는 씨퀀스를 포함하는 DNA단편과 활성제 단백질을 코딩하는 씨퀀스를 포함하는 DNA단편에 단세포생물을 형질전화시키고, (b) 형질전환된 단세포를 적당한 배지에서 배양하는 상기 단계로 구성된 것을 특징으로하는 활성티로시나제의 제조방법.
  40. 제39항에 있어서, 상기 단세포생물이 효모인 것을 특징으로하는 활성티로시나제의 제조방법.
  41. 제39항에 있어서, 상기 단세포생물이 Streptomyces속인 것을 특징으로하는 활성티로시나제의 제조방법.
  42. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019920703432A 1990-06-29 1991-06-28 형질전환된 미생물에 의한 멜라닌의 제조방법 KR0149181B1 (ko)

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