KR970701264A - 지질분해효소의 변이체 제조방법(a method of preparing a viriant of a lipolytic enzyme) - Google Patents

지질분해효소의 변이체 제조방법(a method of preparing a viriant of a lipolytic enzyme)

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KR970701264A
KR970701264A KR1019960704602A KR19960704602A KR970701264A KR 970701264 A KR970701264 A KR 970701264A KR 1019960704602 A KR1019960704602 A KR 1019960704602A KR 19960704602 A KR19960704602 A KR 19960704602A KR 970701264 A KR970701264 A KR 970701264A
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lipase
bacillus
dna sequence
cell
variant
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KR1019960704602A
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스벤트젠 알란
그로트 클라우젠 입
지그르트 오켈스 옌스
텔러젠 마리안
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안네 제케르
노보 노르디스크 아크티에 셀스카브
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Publication of KR970701264A publication Critical patent/KR970701264A/ko

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38663—Stabilised liquid enzyme compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
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    • Y10S435/832—Bacillus
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    • Y10S435/8215—Microorganisms
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    • Y10S435/848—Escherichia
    • Y10S435/849—Escherichia coli
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    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
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    • Y10S435/886—Streptomyces
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    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/913—Aspergillus

Abstract

모 지질분해효소를 제조하는 방법은, (a) 모 지지분해효소를 암호하하는 DNA 서열을 임의적 돌연변이유발을 일으키게 하고, (b) 숙주세포에서 단계 (a)에서 얻은 돌연변이된 DNA 서열을 발현시키고, 및 (c) 모 지질분해효소와 비교하여 칼슘에 대해 감소된 의존성 및/또는 세제나 세제 성분에 대해 개선된 내성을 갖는 돌연변이된 지질분해 효소를 발현하는 숙주세포를 스크리닝하는 것으로 이루어진다.

Description

지질분해효소의 변이체 제조방법(A METHOD OF PREPARING A VIRIANT OF A LIPOLYTIC ENZYME)
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 pYESHL의 제한 지도이고,
제2도는 플라스미드 pA01의 제한 지도이고,
제3도는 플라스미드 pAHL의 제한 지도이고, 및
제4도 및 제5도는 본 발명의 변이체를 암호화하는 유전자의 구조물이다.

Claims (46)

  1. (a) 모 지지분해효소를 암호하하는 DNA 서열을 임의적 돌연변이유발을 일으키게 하고, (b) 숙주세포에서 단계(a)에서 얻은 돌연변이된 DNA 서열을 발현시키고, 및 (c) 모 지질분해효소와 비교하여 칼슘에 대해 감소된 의존성 및/또는 세제나 세제성분에 대해 개선된 내성을 갖는 돌연변이된 지지분해효소를 발현하는 숙주세포를 스크리닝하는 것으로 이루어진 모 지질분해효소의 변이체를 제조하는 방법.
  2. 제1항에 있어서, 임의적 돌연변이 유발은 물리적 또는 화학적 돌연변이유발제의 사용, 올리고뉴클레오티드의 사용 또는 PCR 발생 돌연변이유발제의 사용에 의해 실행되는 것을 특징으로 하는 방법.
  3. 제2항에 있어서, 돌연변이유발제는 포름산, UV 조사, 히드록실아민, N-메틸-N′-니트로-N-니트로소구아니딘(MNNG), O-메틸히드록실아민, 아질산, 에틸메탄술포네이트(EMS), 중아황산나트륨 및 뉴클레오티드 유사체로부터 선택되는 것을 특징으로 하는 방법.
  4. 제1항에 있어서, 돌연변이된 DNA 서열의 발현은 돌연변이된 DNA 서열의 발현을 허용하는 기능을 암호화하는 DNA 서열을 임의로 더 포함하는 돌연변이된 DNA 서열을 갖는 적당한 숙주세포를 형질전환하고 돌연변이된 DNA 서열을 발현하기 위해 적당한 조건하에서 단계(b)에서 얻은 숙주세포를 배양함으로써 실행되는 것을 특징으로 하는 방법.
  5. 제1항에 있어서, 돌연변이된 DNA 서열을 발현하는데 사용된 숙주세포가 미생물 세포인 것을 특징으로 하는 방법.
  6. 제5항에 있어서, 숙주세포가 균류 또는 박테리아 균주의 세포인 것을 특징으로 방법.
  7. 제6항에 있어서, 숙주세포가 A. niger, A. oryzae 및 A. nidulans 같은 Aspergillus속의 세포, 또는 S. cereviciae 같은 Saccharomyces 속의 세포인 것을 특징으로 하는 방법.
  8. 제6항에 있어서, 숙주세포가 그람-양성의 박테리아의 균주의 세포, 예를 들어 Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis 또는 streptomyces lividans 또는 streptomyces murinus 같은 Bacillus 속의 세포, E.coli 같은 그람-음성의 박테리아 균주의 세포인 것을 특징으로 하는 방법.
  9. 제1항에 있어서, 돌연변이된 지지분해효소는 비이온성, 음이온성, 양이온성, 양성이온성 또는 양쪽성의 계면활성제에 대한 개선된 내성을 갖는 것을 특징으로 하는 방법.
  10. 제9항에 있어서, 비이온성 계면활성제는 알코올 에톡실레이트 및/또는 음이온성 계면활성제는 LAS 또는 알킬술페이트인 것을 특징으로 하는 방법.
  11. 제1항에 있어서, 단계(c)에서 스크리닝된 숙주세포가 두번째의 돌연변이유발처리, 재스크리닝, 재분리 및/또는 재클로닝을 받게 하는 것을 특징으로 하는 방법.
  12. 제1항 내지 제11항 중의 어느 한 항에 있어서, 임의적 돌연변이유발이 모 지질분해효소를 암호화하는 DNA 서열의 부분으로 국소화되는 것을 특징으로 하는 방법.
  13. 제1항 내지 제12항 중의 어느 한 항에 있어서, 모 지질분해효소가 리파제, 에스테라제, 쿠티나제 또는 포스포리파제인 것을 특징으로 하는 방법.
  14. 제12항 또는 제13항에 있어서, 모 지질분해효소가 리파제이고 국소화된 임의적 돌연변이유발이 지질 접촉대를 암호화하는 DNA 서열의 부분 또는 모 리파제의 그 부분에서 실행되는 것을 특징으로 하는 방법.
  15. 제14항에 있어서, 국소화된 임의적 돌연변이유발이 리드 부위를 암호화하는 DNA 서열 및/또는 모리파제의 소수성의 갈라진 틈의 부분, 또는 상기 리드 부위 및/또는 소수성 결합 갈라진 틈의 부분에서 실행되는 것을 특징으로 하는 방법.
  16. 제1항 내지 제13항 중의 어느 한 항에 있어서, 모 지질분해효소가 미생물로부터 유도될 수 있는 것을 특징으로 하는 방법.
  17. 제18항에 있어서, 모 지질분해효소가 균류에서 유도될 수 있는 것을 특징으로 하는 방법.
  18. 제17항에 있어서, 모 지질분해효소가 암호화하는 DNA 서열이 Humicola sp., Rhizomucor sp., Rhizopus sp., Candida sp.의 균주로부터 유도될 수 있는 것을 특징으로 하는 방법.
  19. 제18항에 있어서, 모 지질분해효소가 리파제이고 모리파제를 암호화하는 DNA 서열이 H. lanuginosa 균주, 예를 들면, H. lanuginosa 균주 DSM 4109, Rh. mucor의 균주, 또는 C. antarctica의 균주로부터 유도될 수 있는 것을 특징으로 하는 방법.
  20. 제19항에 있어서, 임의적 돌연변이를 일으키는 DNA 서열이 DSM 4109로부터 얻을 수 있는 H lanuginosa 리파제의 아미노산 잔기 21∼27, 56∼64, 81∼99, 108∼116, 145∼147, 202∼213, 226∼227, 246∼259 또는 263∼269에 의해 정의된 부위중 적어도 한 곳을 암호화하는 것을 특징으로 하는 방법.
  21. 제20항에 있어서, 국소화된 임의적 돌연변이는 상기 부위의 적어도 두 곳에서 실행되는 것을 특징으로 하는 방법.
  22. 제16항에 있어서, 모 지질분해효소가 박테리아에서 유도될 수 있는 것을 특징으로 하는 방법.
  23. 제22항에 있어서, 모 지질분해효소를 암호화하는 DNA P. cepacia, P. alcaligenes, P. pseudoalcaligens 또는 P. fragi 같은 Pseudomonas spp.의 균주, 또는 Bacillus의 균주에서 유도될 수 있는 것을 특징으로 하는 방법.
  24. 제1항 내지 23항 중의 어느 한 항을 따른 방법에 의해 제조된 지지분해효소의 변이체.
  25. 제24항에 있어서, 다음의 위치는 적어도 한 곳에서의 돌연변이로 이루어지는 DSM 4109로부터 얻을 수 있는 H. lanuginosa 리파제 또는 그의 유사체의 변이체인 것을 특징으로 하는 변이체: K46, E56, S58, G61, T64, N73, S83, I90, G91, N92, N94, D96, L97, K98, E99, I100, D102, A121, E129, D167, R205, E210, K237, N251, I252, D254, P256, G263, L264 또는 T267.
  26. 아미노산 잔기 56∼64, 83∼100 또는 205∼211에 의해 정의된 부위중 적어도 한 곳에서의 돌연변이를 지니는, 균주 DSM 4109로부터 얻을 수 있는 H. lanuginosa 리파제 또는 상기 리파제의 유사체의 변이체.
  27. 제26항에 있어서, 다음의 돌연변이중 적어도 하나로 이루어지는 것을 특징으로 하는 변이체: K46R, D57G, S58F, G61S, D62C, T64R, S83T, I90F, G91A, N92H, N94I, N94K, L97M, K98I, I100V, D102K, A121V, E129K, D167G, R205K, E210W, K237M, N259W, I252L, D254W, P256T, G263A, L264Q 또는 T267W.
  28. 다음의 돌연변이중 적어도 하나로 이루어지는 DSM 4109로부터 얻을 수 있는 H. lanuginosa 리파제 또는 그의 유사체의 변이체: n87K+D96V, E87K+G91A+D96A, N94K+F95L+D96H, A121V+R205K+E210Q, F95C+D96N, G91S+L93V+F95C, E87K+G91A+D96R+I100V, E87K+G91A, S83T+E87K+Q249R, S83T+E87K+W89G+G91A+N94K+D96V, N73D+S85T+E87K+G91A+N94K+D94A, E87K+G91A+L93I+N94K+D96A, D167G+E210V, N73D+E87K+G91A+N94I+D96G, S83T+E87K+G91A+N92H+N94K+D96M, E210W, E56T+D57L+I90F+D96L+E99K, E56R+D57L+V60M+D62N+S83T+D96P+D102E, D57G+N94K+K96L+L97M, E87K+G91A+D96R+I100V+E129K+K237M+I252L+P256T+G263A+L264Q, E56R+D57G+S58F+D62C+T64R+E87G+G91A+F95L+D96P+K98I+K237M, K46R, E56R, G61S, D102K, D167F, N73D+E87K+G91A+N94I+D96F, E210V, E210W, N251W+D254W+T267W, S83T+E87K+G91A+N92H+N94K+D96M, E56R+I90F+D96L+E99K, D57G+N94K+D96L+L97M.
  29. 제1항 내지 제23항 중의 어느 항에 따르는 방법에 단계(c)에서 스크리닝된 숙주세포부터 분리되는, 모 지질분해효소와 비교하여 칼슘에 대해 감소된 의존성 및/또는 세제나 세제 성분에 대해 개선된 내성을 갖는 지질분해효소의 변이체를 암호화하는 돌연변이된 DNA 서열로 이루어지는 DNA 구조물.
  30. 제24항 내지 제28항 중의 어느 항에 따르는 H.lanuginosa 리파제 변이체를 암호화하는 것을 특징으로 하는 DNA 구조물.
  31. 제29항 또는 제30항에 따른 DNA 구조물을 분비하는 벡터.
  32. 제31항에 있어서, 플라스미드 또는 박테리오파지인 것을 특징으로 하는 벡터.
  33. 제31항 또는 제32항에 있어서, 모 지질분해효소의 변이체의 발현을 허용하는 DNA 서열을 더 포함하는 발현 벡터인 것을 특징으로 하는 벡터.
  34. 제29항 또는 제30항에 따르는 DNA 구조물 또는 제31항 내지 제33항 중의 어느 항을 따른 벡터를 분비하는 숙주세포.
  35. 제34항에 있어서, 미생물 세포인 것을 특징으로 하는 세포.
  36. 제35항에 있어서, 균류 또는 박테리아 균주의 세포인 것을 특징으로 하는 세포.
  37. 제36항에 있어서, A. niger, A. oryzae 및 A. nidulans 같은 Aspergillus 속의 세포, 또는 S. cereviciae 같은 Saccharomyces 속의 세포인 것을 특징으로 하는 세포.
  38. 제36항에 있어서, 그람-양성의 박테리아 균주의 세포, 예를들어, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis 또는 streptomyces lividans 또는 streptomyces murinus 같은 Bacillus 속의 세포, 또는 E.coli 같은 그람-음성의 박테리아 균주의 세포인 것을 특징으로 방법.
  39. 제1항 내지 제23항 중의 어느 항의 방법에 따라 변이체 지질분해효소를 제조하고 단계(c)에서 스크리닝된 숙주세포로부터 지질분해효소 변이체를 회수하는 것으로 이루어지는, 모 지질분해효소와 비교하여 칼슘에 대해 감소된 의존성 및/또는 세제나 세제 성분에 대해 개선된 내성을 갖는 모 지질분해효소의 변이체를 제조하는 방법.
  40. 변이체를 발현하기에 적당한 조건하에서 제34항 내지 제38항 중의 어느 항에 따른 숙주세포를 배양하고 배양물로부터 발현된 변이체를 회수하는 것으로 이루어지는 모 지질분해효소와 비교하여 칼슘에 대해 감소된 의존성 및/또는 세제나 세세 성분에 대해 개선된 내성을 갖는 모 지질분해효소의 변이체를 제조하는 방법.
  41. 제39항 또는 제40항에 따른 방법에 의해 제조된 지질분해효소의 변이체.
  42. 임의로 비-분진성 과립, 안정화된 액체 또는 보호된 효소의 형태로, 제24항 내지 제28항 또는 제41항 중의 어느 항에 따른 지질분해효소의 변이체로 이루어지는 세제 첨가제.
  43. 제42항에 있어서, 첨가제 g당 효소 단백질 0.02∼200㎎을 함유하는 것을 특징으로 하는 세제 첨가제.
  44. 제42항 또는 제43항에 있어서, 프로테아제, 아밀라제, 피옥시다제, 쿠티나제, 리파제 및/또는 셀룰라제 같은 또다른 효소를 추가로 포함하는 것을 특징으로 하는 세제 첨가제.
  45. 제24항 내지 제28항 중의 어느 한 항 또는 제41항 중에 따른 지지분해효소의 변이체로 이루어지는 세제 조성물.
  46. 제45항에 있어서, 추가로 프로테아제, 아밀라제, 피옥시다제, 쿠티나제, 리파제 및/또는 셀룰라제 같은 또다른 효소를 포함하는 것을 특징으로 하는 세제 조성물.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019960704602A 1994-02-22 1995-02-22 지질분해효소의 변이체 제조방법(a method of preparing a viriant of a lipolytic enzyme) KR970701264A (ko)

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