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KR930702514A - 리파제 변체 - Google Patents

리파제 변체

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KR930702514A
KR930702514A KR1019930700773A KR930700773A KR930702514A KR 930702514 A KR930702514 A KR 930702514A KR 1019930700773 A KR1019930700773 A KR 1019930700773A KR 930700773 A KR930700773 A KR 930700773A KR 930702514 A KR930702514 A KR 930702514A
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lipase
lipase variant
amino acid
variant
porridge
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알란 스벤드센
입 그로트 클라우젠
샴칸트 아난트 파트카아
에릭 고름센
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안네 제케르
노보 노르디스크 아크티에 셀스카브
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Priority claimed from DK219590A external-priority patent/DK219590D0/da
Priority claimed from DK219690A external-priority patent/DK219690D0/da
Application filed by 안네 제케르, 노보 노르디스크 아크티에 셀스카브 filed Critical 안네 제케르
Publication of KR930702514A publication Critical patent/KR930702514A/ko

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Abstract

포켓이 지질접촉 부위의 부분을 형성하고 지절접촉부에 의해 둘러싸인 리파제 분자의 우세하게 소수성인 연장된 결합포켓내에 위치된 활성세린을 포함하는 트립신-같은 촉매 3체를 함유하는 리파제가 지질접촉부의 정전기적 전하 및/또는 소수성을 변화시키기 위해서 또는 리파제의 지질접촉부의 표면형태를 변화시키기 위해서 지질접촉부내의 하나 이상의 아미노산 잔기의 삭제 또는 치환에 의해 돌연변이 되었다.

Description

리파제 변체
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1A 및 B도는 리파제가 각기 불활성(A) 및 활성(B)형태일때 H·lanuginosa 리파제의 지질접촉부의 3-차원 구조를 나타내는 컴퓨터 모델이다, 제2A 및 2B도는 리파제가 각기 불황성(A) 및 활성(B)형태일때 Rh·miehei 리파제의 지질접촉부의 3-차원 구조를 나타내는 컴퓨터 모델이다.

Claims (161)

  1. 리파제 분자의 우세하게 소수성인 연장된 결합포켓내에 위치된 활성세린을 포함하는 트립신-같은 촉매 3체를 함유하는 모(母) 리파제의 리파제 변체에 있어서, 가수분해시 또는 가수분해 동안 기질과의 상호작용에 참여할 수 있는 활성세린 잔기를 포함하는 리파제 구조부내에 위치된 잔기들을 함유하는 지질 접촉부의 정전기적 전하 및/또는 소수성이 하나 이상의 음전하 아미노산잔기를 중성의 또는 양전하의 아미노산잔기(들)에 의해 치환 또는 삭제함에 의하여, 및/또는 하나 이상의 중성의 아미노산 잔기를 양전하 잔기를 소수성 아미노산잔기(들)에 의해 치환 또는 삭제함에 의하여 변화되는 것을 특징으로 하는 리파제 변체.
  2. 제1항에 있어서, 상기의 지질 접촉부의 하나 이상의 글루탐산, 아스파르트산 잔기가 글루타민, 아스파라긴, 알라닌, 류신, 발린, 세린, 트레오닌, 리신, 또는 아르기닌에 의해 치환되는 것을 특징으로 하는 리파제 변체.
  3. 제1항 내지 제2항중 어느 하나에 있어서, 모 리파제가 미생물의 리파제인 것을 특징으로 하는 리파제 변체.
  4. 제3항에 있어서, 모 리파제가 곰팡이의 리파제인 것을 특징으로 하는 리파제 변체.
  5. 제4항에 있어서, 모 리파제가 Humicola, 또는 Rhizomucor 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  6. 제5항에 있어서, 모 리파제가 Rhizomucor miehei 리파제인 것을 특징으로 하는 리파제 변체.
  7. 제6항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이, 치환되는 것을 특징으로 하는 리파제 변체 :
  8. D 91N, K, R, A, V, L, S, T,
  9. D256N, K, R, A, V, L, S, T,
  10. D226N, K, R, A, V, L, S, T,
  11. D 61N, K, R, A, V, L, S, T,
  12. D113N, K, R, A, V, L, S, T,
  13. E201Q, K, R, A, V, L, S, T,
  14. D243N, K, R, A, V, L, S, T.
  15. 제5항에 있어서, 모 리파제가Humicola Rhizomucor리파제인 것을 특징으로 하는 리파제 변체.
  16. 제8항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  17. E 87Q, K, R, A, N, T, S, L, V,
  18. D254N, K, R, A, N, T, S, L, V,
  19. D242N, K, R, A, N, T, S, L, V,
  20. E210Q, K, R, A, N, T, S, L, V,
  21. E 56Q, K, R, A, N, T, S, L, V,
  22. D 96N, K, R, A, N, T, S, L, V,
  23. D111N, K, R, A, N, T, S, L, V,
  24. D 62A, Q, N, T, S, K, R, L, V,
  25. E219A, Q, N, T, S, K, R, L, V,
  26. E234A, Q, N, T, S, K, R, L, V,
  27. E 57A, Q, N, T, S, K, R, L, V,
  28. E 99A, Q, N, T, S, K, R, L, V,
  29. D 27A, Q, N, T, S, K, R, L, V, 또는
  30. E239A, Q, N, T, S, K, R, L, V.
  31. 제9항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  32. E87Q+D254N+D242N+E210Q,
  33. E87Q+D254N+E210Q,
  34. E96N+E87Q+D254N,
  35. R209A+E210A.
  36. 제8항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  37. T267K, R,
  38. S 85K, R,
  39. T226K, R,
  40. N 88K, R,
  41. N 92K, R,
  42. I255K, R,
  43. I202K, R,
  44. L206K, R,
  45. L259K, R,
  46. V202K, R, 또는
  47. L227K, R
  48. 제4항에 있어서, 모 리파제가 효모 리파제인 것을 특징으로 하는 리파제 변체.
  49. 제12항에 있어서, 모 리파제가 Candida 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  50. 제3항에 있어서, 모 리파제가 세균의 리파제인 것을 특징으로 하는 리파제 변체.
  51. 제14항에 있어서, 모 리파제가 Pseudomonas 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  52. 리파제 분자의 우세하게 소수성인 연장된 결합포켓내에 위치된 활성세린을 포함하는 트립신-같은 촉매 3체를 함유하는 리파제 변체에 있어서, 상기의 리파제 변체가 지질 접촉부의 표면형태를 변화시키기 위해서 가수분해시 또는 가수분해 동안 기질과의 상호작용에 참여하는 활성세린 잔기를 포함하는 리파제 구조부내에 위치된 잔가딜을 함유하는 지질 접촉부의 서열을 구성하는 하나 이상의 아미노산 잔기의 위치에 하나 이상의 아미노산 잔기가 치환, 삭제, 또는 삽입된 것을 특징으로 하는 리파제 변체.
  53. 제16항에 있어서, 하나 이상의 아미노산 잔기가 하나 이상의 다른 덜 부피있는 아미노산 잔기에 의하여 치환된 것을 특징으로 하는 리파제 변체.
  54. 제17항에 있어서, 하나 이상의 아미노산 잔기가 발린, 트레오닌, 세린, 글리신, 또는 알라닌에 의해 치환된 것을 특징으로 하는 리파제 변체.
  55. 제16항에 있어서, 지질 접촉부내의 고리서열부를 형성하는 하나 이상의 아미노산 잔기가 삭제된 것을 특징으로 하는 리파제 변체.
  56. 제19항에 있어서, 2-8, 특히 2-6 아미노산 잔기가 지질 접촉부내의 하나 이상의 고리서열로 부터 삭제된 것을 특징으로 하는 리파제 변체.
  57. 제16항 내지 제20항중 어느 하나에 있어서, 모 리파제가 미생물의 리파제인 것을 특징으로 하는 리파제 변체.
  58. 제21항에 있어서, 모 리파제가 곰팡이의 리파제인 것을 특징으로 하는 리파제 변체.
  59. 제22항에 있어서, 모 리파제가 Humicola 또는 Rhizomucor 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  60. 제23항에 있어서, 모 리파제가 Rhizomucor miehei 리파제인 것을 특징으로 하는 리파제 변체.
  61. 제24항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  62. I204V, A, T, S, G,
  63. L208V, A, T, S, G,
  64. F213V, A, T, S, G, 또는
  65. I254V, A, T, S, G.
  66. 제24항에 있어서, 하나 이상의 아미노산 잔기가 하기의 위치중 하나 이상에서 삭제된 것을 특징으로 하는 리파제 변체 :
  67. 82-113, 211-215, 235-243, 245-269 또는 264-269
  68. 제26항에 있어서, 하기와 같이 변형된 것을 특징으로 하는 리파제 변체 :
  69. N264*+T265*+G266*+L267*+C268*+T269*+C22T.
  70. 제26항에 있어서, 하기의 아미노산 잔기가 삭제된 것을 특징으로 하는 리파제 변체 :
  71. F213*+F215*
  72. D238*+L239*+E240*+D243*, 또는
  73. F251*+T252*+S247*
  74. 제23항에 있어서, 모 리파제가Humicola lanuginosa리파제인 것을 특징으로 하는 리파제 변체.
  75. 제29항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  76. I202V, A, T, S, G,
  77. L206V, A, T, S, G,
  78. F211V, A, T, S, G, 또는
  79. I255V, A, T, S, G.
  80. 제29항에 있어서, 하나 이상의 아미노산 잔기가 하기의 위치중 하나 이상에서 삭제된 것을 특징으로 하는 리파제 변체 :
  81. 82-112, 209-213, 238-245, 247-254 또는 264-269.
  82. 제31항에 있어서, 하기와 같이 변형된 것을 특징으로 하는 리파제 변체 :
  83. N264*+T265*+G266*+L267*+C268*+T269*+C22T.
  84. 제31항에 있어서, 하기의 아미노산 잔기가 삭제된 것을 특징으로 하는 리파제 변체 :
  85. R209*+E210*
  86. F211*+Y213*
  87. D242*+E239*+I241, 또는
  88. N247*+D254*
  89. 제21항에 있어서, 모 리파제가 효모 리파제인 것을 특징으로 하는 리파제 변체.
  90. 제34항에 있어서, 모 리파제가 Candida 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  91. 제21항에 있어서, 모 리파제가 세균의 리파제인 것을 특징으로 하는 리파제 변체.
  92. 제36항에 있어서, 모 리파제가Pseudomonas균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  93. (i)리파제 분자의 우세하게 소수성인 연장된 결합포켓내에 위치된 활성세린을 포함하는 트립신-같은 촉매 3체 및 (ii)리파제가 불활성 형태일때 활성세린을 덮고 리파제가 활성세린을 지질 기질에 접근가능하게 만들기 위해 활성화될때 그것의 형태를 변화시키는 표면고리구조, 여기서 고리구조는 결합포켓에 면한 우세하게 소수성인 안쪽 표면과 우세하게 친수성인 바깥쪽 표면을 가지는 것을 함유하는 유형의 리파제 변체에 있어서, 상기의 리파제 변체가 가수분해시 또는 가수분해 동안 기질과의 상호작용 또는 표면고리구조를 이동시키는 것에 참여하는 활성세린 잔기를 포함하는 리파제 구조부내에 위치된 잔기들을 함유하는 지질 접촉부 서열을 구성하는 및/또는 고리구조의 서열을 구성하는 하나 이상의 아미노산 잔기의 위치에 하나 이상의 아미노산 잔기가 치환, 삭제, 또는 삽입된 것을 특징으로 하는 리파제 변체.
  94. 제38항에 있어서, 고리구조의 적어도 하나의 아미노산 잔기가 시스테인에 의해 치환되고, 및 적어도 하나의 다른 아미노산 잔기가 시스테인에 의해 치환되고, 두시스테인 잔기가 서로서로에 대해 이황화물 결합을 형성하도록 위치되는 것을 특징으로 하는 라파제 변체.
  95. 제38항 내지 제39항중 있어서, 모 리파제가 미생물의 리파제인 것을 특징으로 하는 리파제 변체.
  96. 제38항 내지 제40항중 어느 하나에 있어서, 모 리파제가 곰팡이의 리파제인 것을 특징으로 하는 리파제 변체.
  97. 제41항에 있어서, 모 리파제가 Humicola, 또는 Rhizomucor 균주로 부터 추출되는 것을 특징으로 하는 리파제 변체.
  98. 제42항에 있어서, 모 리파제가 Rhizomucor miehei 리파제인 것을 특징으로 하는 리파제 변체.
  99. 제43항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  100. S114C+A90C
  101. R 86C+D61C,
  102. S 84C+D61C,
  103. N 74C+D61C,
  104. Y 60C+R78C, 또는
  105. Y 60C+B87C,
  106. 제43항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이 치환되는 것을 특징으로 하는 리파제 변체 :
  107. I240V, T, S, A, G,
  108. L208V, T, S, A, G,
  109. V254V, T, S, A, G,
  110. L255V, T, S, A, G,
  111. L258V, T, S, A, G,
  112. L267V, T, S, A, G,
  113. F 94L, T, K, 또는
  114. F213L, T, K
  115. 제43항에 있어서, 하기의 아미노산 치환을 포함하는 것을 특징으로 하는 리파제 변체 :
  116. I204T+ :255T+L257T, 또는
  117. L208T+ V254T+L258T
  118. 제42항에 있어서, 모 리파제가 Humicola, 또는 lanuginosa 리파제인 것을 특징으로 하는 리파제 변체.
  119. 제47항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이, 치환되는 것을 특징으로 하는 리파제 변체:
  120. G 61C+N88C,
  121. G 61C+E87C,
  122. D 62C+E87C,
  123. D 62C+S85C,
  124. D 62C+N88C, 또는
  125. S116C+G91C
  126. 제47항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이, 치환되는 것을 특징으로 하는 리파제 변체:
  127. L 93V, T, S, A, G,
  128. I 90V, T, S, A, G,
  129. I 86V, T, S, A, G,
  130. I202V, T, S, A, G,
  131. I206V, T, S, A, G,
  132. I255V, T, S, A, G,
  133. L259V, T, S, A, G,
  134. I265V, T, S, A, G,
  135. F 95L, T, K, 또는
  136. F211L, T, K,
  137. 제49항에 있어서, 하나 이상의 아미노산 잔기가 하기와 같이, 치환되는 것을 특징으로 하는 리파제 변체:
  138. F 95K,
  139. I 86T,
  140. I 90T,
  141. I255T,
  142. L259T,
  143. L206T, 또는
  144. L206T+I255T+L259T
  145. 제40항에 있어서, 모 리파제가 효모 리파제인 것을 특징으로 하는 리파제 변체.
  146. 제51항에 있어서, 모 리파제가Candida균주로 부터 추출되는 것을 특징으로 하는 리파제 변체:
  147. 제40항에 있어서, 모 리파제가 세균의 리파제인 것을 특징으로 하는 리파제 변체:
  148. 제53항에 있어서, 모 리파제가Pseudomonas균주로 부터 추출되는 것을 특징으로 하는 리파제 변체:
  149. 제1항 내지 제54항중 어느 하나에 따른 리파제 변체를 암호화하는 DNA 서열을 포함하는 DNA 구조체.
  150. 제55항에 따른 DNA 구조체를 운반하는 재조합 발현벡터.
  151. 제55항에 따른 DNA 구조체를 운반하는 재조합 발현벡터.
  152. 제57항에 있어서, 곰팡이, 세포, 예컨대,A.niger,A.oryzae또는A.nidulans따위의 속Aspergillus에 속하는 곰팡이세포, 효모세포, 예컨대S.cerevisiae따위의Saccharomyces균주에 속하는 효모세포, 또는H.polymorpha따위의 속Hansenula의 또는P.pastoris따위의 속Phichia의 메틸로트로픽 효모(methylotrophic yeast), 또는 세균세포, 예컨대B.subtilis,B.lentus따위의Bacillus균주에 속하는 세균 세포인 것을 특징으로 하는 세포.
  153. 제57항에 있어서, 식물세포, 예컨대Solamun tuberosum, 또는Nicotiana tabacum따위의Solanaceae에 속하는 식물세포인 것을 특징으로 하는 세포.
  154. 제59항에 따른 세포를 함유하는 식물.
  155. 제1항 내지 제54항중 어느 하나에 따른 리파제 변체의 제조방법에서, 제57항 내지 제59항중 어느 하나에 따른 세포, 또는 제60항에 따른 식물이 리파제 변체의 제조에 도움이 되는 조건하에서 배양 또는 생장되고 그 다음에 리파제 변체가 배양물 또는 식물체로 부터 회수되는 것을 특징으로 하는 리파제 변체 제조방법.
  156. 제1항 내지 제54항중 어느 하나에 따른 리파제 변체를 포함하는 세정제 첨가물에서, 선택적으로 비-살포성 과립, 안정화된 액체 또는 보호된 효소의 형태의 세정제 첨가물.
  157. 제62항에 있어서, 첨가물 g당 효소 단백질을 0.02-200mg 포함하는 것을 특징으로 하는 세정제 첨가물.
  158. 제62항 또는 제63항에 있어서, 부가적으로 프로테아제, 아밀라제, 퍼옥시다제 및/또는 셀룰라제 따위의 다른 효소를 포함하는 것을 특징으로 하는 세정제 첨가물.
  159. 제1항 내지 54항중 어느 하나에 따른 리파제 변체를 함유하는 세정제 조성물.
  160. 제65항에 있어서, 부가적으로 프로테아제, 아밀라제, 퍼옥시다제 및/또는 셀룰라제 따위의 다른 효소를 포함하는 것을 특징으로 하는 세정제 조성물.
  161. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019930700773A 1990-09-13 1991-09-13 리파제 변체 KR930702514A (ko)

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DK219490A DK219490D0 (da) 1990-09-13 1990-09-13 Enzymmutanter
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DK2195/90 1990-09-13
DK219590A DK219590D0 (da) 1990-09-13 1990-09-13 Lipasemutanter
DK219690A DK219690D0 (da) 1990-09-13 1990-09-13 Lipasevarianter
PCT/DK1991/000271 WO1992005249A1 (en) 1990-09-13 1991-09-13 Lipase variants

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DE69129988D1 (de) 1998-09-17
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AU8617291A (en) 1992-04-15
ES2121786T3 (es) 1998-12-16
WO1992005249A1 (en) 1992-04-02
JP3469234B2 (ja) 2003-11-25
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FI120044B (fi) 2009-06-15
EP0548228A1 (en) 1993-06-30

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