KR20080045766A - 비피도박테륨에서 단리한 베타-갈락토시다제 - Google Patents
비피도박테륨에서 단리한 베타-갈락토시다제 Download PDFInfo
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- KR20080045766A KR20080045766A KR1020087010589A KR20087010589A KR20080045766A KR 20080045766 A KR20080045766 A KR 20080045766A KR 1020087010589 A KR1020087010589 A KR 1020087010589A KR 20087010589 A KR20087010589 A KR 20087010589A KR 20080045766 A KR20080045766 A KR 20080045766A
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Abstract
본 발명은 비피도박테륨 비피둠에서 단리한, 트랜스갈락토실화 활성을 보유하는 β-갈락토시다제 및 β-갈락토시다제 단백질의 C-말단 종결부가 결실되어 트랜스갈락토실화 활성이 히드롤라제 활성 보다 더 높은 말단절단형 효소에 관한 것이다. 트랜스갈락토실라제 활성은 기질로서 락토스를 사용하는 경우, 갈락토-올리고당류를 생산한다. 갈락토-올리고당류는 낙농업에서 수많은 용도로 사용할 수 있는 건강 촉진 비피도박테륨의 성장을 향상시킨다.
비피도박테륨 비피둠, β-갈락토시다제, 트랜스갈락토실라제 활성, 갈락토-올리고당류
Description
본 발명은 발효시킨 유제품의 개선에 관한 것이다. 특히, 본 발명은 트랜스갈락토실화 활성을 보유하는 β-갈락토시다제에 관한 것이다. 더욱 특히, 본 발명은 비피도박테륨 비피둠 (Bifidobacterium bifidum)에서 단리한 β-갈락토시다제로서 상기 단백질의 C-말단 종결부가 결실되어 트랜스갈락토실화 활성이 히드롤라제 활성 보다 더 높은 말단절단형 (truncated) 효소로 생성된 β-갈락토시다제에 관한 것이다. 트랜스갈락토실라제 활성은 기질로서 락토스를 사용하는 경우, 갈락토-올리고당류를 생산한다. 갈락토-올리고당류는 낙농업에서 수많은 용도로 사용할 수 있는 건강 촉진 비피도박테륨의 성장을 향상시킨다.
비피도박테륨 속은 유제품 산업에서 다양한 유제품 발효에 가장 널리 이용되는 유형의 박테리아 배양물 중 하나이다. 또한, 비피도박테륨을 함유하는 제품의 섭취는 건강을 촉진하는 효과가 있다. 이러한 효과는 장내 내용물의 pH 감소에 의해서만이 아니라, 항생제 복용 등으로 인해 장내 플로라 (flora)가 파괴된 개인에서 장내 플로라를 복구할 수 있는 비피도박테륨의 능력에 의해서도 달성된다. 또 한, 비피도박테륨은 잠재적으로 해로운 장내 미생물들을 배출시키는 (outcompeting) 능력도 보유한다.
갈락토-올리고당류는 비피도박테륨의 성장을 향상시킨다고 공지되어 있다. 이러한 효과는 갈락토-올리고당류를 탄소 공급원으로 활용하는 비피도박테륨의 독특한 능력을 통해 달성되는 것으로 보인다. 또한, 식이보조제로서의 갈락토-올리고당류는 수많은 장기간의 질환을 보호하는 효과를 갖는 것으로 여겨진다. 예를 들어, 갈락토-올리고당류의 복용은 래트에서의 직장결장암 진행에 대한 고도의 보호 효과를 나타내는 것으로 밝혀진 바 있다 [Wijnands, et al., 1999]. 그러므로, 식이보조제 및 유제품을 개선하는 산업에 사용하기 위해서, 갈락토-올리고당류를 생산하는 저렴하고 효율적인 방법의 개발에 많은 관심이 있다.
효소 β-갈락토시다제 (EC 3.2.1.23)는 통상적으로 락토스를 단당류인 D-글루코스 및 D-갈락토스로 가수분해한다. β-갈락토시다제의 정상적인 효소 반응에서, 상기 효소는 락토스를 가수분해하고 갈락토스 단당류를 갈락토스-효소 복합체 중에서 일시적으로 결합하였다가 상기 갈락토스를 물의 히드록실기에 전달함으로써, D-갈락토스 및 D-글루코스를 유리시킨다. 그러나, 락토스가 고농도인 경우, 일부의 β-갈락토시다제는 트랜스갈락토실화라고 지칭되는 과정을 통해 갈락토스를 D-갈락토스 또는 D-글루코스의 히드록실기에 전달하여 갈락토-올리고당류를 생산할 수 있다.
트랜스갈락토실화할 수 있는 효소는 박테리아 및 효모 등을 비롯한 광범위한 범위의 미생물로부터 단리되어 왔다. 갈락토-올리고당류가 건강 촉진 비피도박테 륨의 성장을 향상시킨다는 관찰이 비피도박테륨 및 이의 β-갈락토시다제 효소에 대한 연구를 자극시켰다. 비피도박테륨 브레브 (Bifidobacterium breve)의 β-갈락토시다제 유전자 및 비피도박테륨 롱검 (Bifidobacterium longum)의 β-갈락토시다제 유전자 2가지의 DNA 서열이 진뱅크 (수탁 번호: 각각 E5040 및 AJ242596)에 기탁되어 있다. 문헌 [Dumortier et al. (1994)]에는 비피도박테륨 비피둠 DSM 20215이 3종의 β-갈락토시다제를 함유하고, 이들 효소 중 하나가 트랜스-갈락토실화 성질을 보유한다고 보고되어 있다. 그러나, 이러한 활성을 보유하는 상기 효소를 확인하거나, 상기 효소의 임의의 서열 또는 비피도박테륨 비피둠 DSM 20215로부터의 상응하는 유전자가 공개된 적은 없었다.
β-갈락토시다제를 사용하여 갈락토-올리고당류를 생산하는 방법이 여러 문헌에 보고되어 있다. 예를 들어, 대장균의 β-갈락토시다제는 락토스가 고농도 (0.5 M 락토스 또는 대략 20% 락토스)인 경우에 올리고당류를 생산하는 것으로 나타났다 [Huber et al. 1976]. 각종 호열성 미생물이 고온 및 고농도의 락토스 조건에서 올리고당류를 생산하는 것으로 나타난 바 있는데, 예를 들어 스테리그마토마이세스 엘비애 (Sterigmatomyces elviae)는 60℃에서 20% 락토스로부터 39% 올리고당류를 생산할 수 있으며 [Onishi & Tanaka, 1995], 사카로폴리스포라 렉티비르굴라 (Saccharopolyspora rectivirgula)는 70℃에서 1.75 M 락토스 중에서 41% 올리고당류를 합성할 수 있다 [Nako et al., 1994].
그러나, 상기에서 기재한 효소들은 모두 유의한 트랜스갈락토실라제 활성을 발휘하기 위해서는 고온 또는 고농도의 락토스가 필요하거나, 또는 이들 두가지가 모두 필요하다는 결점이 있다. 그러므로, 산업에 사용하기 위한 갈락토-올리고당류를 생산하는 더욱 저렴하고 더욱 효율적인 방법의 개발이 요구된다.
발명의 요약
본 발명은 비피도박테륨 비피둠의 신규 β-갈락토시다제를 기재한다. 놀랍게도, 상기 효소의 말단절단형 버전은 높은 트랜스갈락토실화 활성을 보유하는 것으로 나타났다. 상기 말단절단형 효소 또는 재조합 말단절단형 효소를 발현하는 숙주 세포를 락토스와 함께 적절한 조건하에서 인큐베이션시키는 경우, 갈락토-올리고당류가 고효율로 생산된다. 유제품 또는 기타의 식품 중 갈락토-올리고당류의 존재는 상기 제품 중 및(또는) 상기 제품을 복용한 소비자의 장내 플로라 중의 건강 촉진 비피도박테륨의 성장을 향상시킨다는 장점이 있다.
본 발명의 제1 측면은 비피도박테륨 비피둠으로부터 단리하여 특성화한 신규 β-갈락토시다제인 OLGA5 (서열 1 및 서열 2)에 관한 것이다. 비피도박테륨 비피둠으로부터의 염색체 DNA를 PstI로 소화시킨 삽입물을 함유하는 플라스미드로 대장균 세포를 형질전환시켰다. 발색성 β-갈락토시다제 기질을 함유하는 플레이트상에서 β-갈락토시다제 활성을 보유한 클론을 선별했다. 양성 콜로니 중 하나는 대략 20 kb의 인서트인 pOLGA5 (서열 1)가 있는 플라스미드를 함유했다. DNA 서열결정을 통해 OLGA5 β-갈락토시다제의 추정되는 아미노산 서열 (서열 2)이 현재 공지된 β-갈락토시다제 길이의 대략 2배이며, 공지된 β-갈락토시다제와의 서열 상동성 정도가 놀라울 만큼 낮다는 것을 밝혀냈다. 대장균 내 재조합 OLGA5의 발현을 통해 상기 효소가 락토스 가수분해 활성 뿐 아니라 트랜스갈락토실화 활성까지 보유한다는 것을 밝혀냈다. OLGA5 효소의 C-말단부는 공지된 β-갈락토시다제와 상동성이 없었다. 이어서, OLGA5의 다양한 C-말단 결실 돌연변이체를 제작하고, 생성된 효소의 가수분해 활성 및 트랜스갈락토실화 활성에 대해 조사했다.
본 발명의 제2 측면은 OLGA5의 결실 돌연변이체, 예를 들어 OLGA347에 관한 것이다. 여러 C-말단 결실 돌연변이체 중에서, 578개 아미노산의 C-말단이 결실된 OLGA347이 비교적 저농도의 락토스와 인큐베이션시키는 경우일지라도 생산되는 올리고당류의 수준은 가장 두드러지게 증가된 것으로 밝혀졌다. 명백하게, 상기 효소는 실제로 모든 갈락토스 분자를 갈락토스 또는 글루코스에 전달한다. 그러므로, OLGA5의 C-말단 종결부의 결실은 상기 효소를 가수분해성 OLGA5 β-갈락토시다제에서 트랜스갈락토실화 OLGA347-트랜스갈락토시다제로 전환시킨다. 천연 OLGA5 효소를 포함하는 다른 트랜스갈락토실화 β-갈락토시다제와는 달리, 상기 말단절단형 β-갈락토시다제 OLGA347은 시험된 모든 락토스 농도에서 갈락토스를 수용자 당 분자에 높은 빈도로 전달한다.
본 발명은 비피도박테륨 비피둠의 신규 β-갈락토시다제를 기재한다. 놀랍게도, 상기 효소의 말단절단형 버전은 높은 트랜스갈락토실화 활성을 보유하는 것으로 나타났다. 상기 말단절단형 효소 또는 재조합 말단절단형 효소를 발현하는 숙주 세포를 락토스와 함께 적절한 조건하에서 인큐베이션시키는 경우, 갈락토-올리고당류가 고효율로 생산된다. 유제품 또는 기타의 식품 중 갈락토-올리고당류의 존재는 상기 제품 중 및(또는) 상기 제품을 복용한 소비자의 장내 플로라 중의 건강 촉진 비피도박테륨의 성장을 향상시킨다는 장점이 있다.
본 발명의 제1 측면은 비피도박테륨 비피둠으로부터 단리하여 특성화한 신규 β-갈락토시다제인 OLGA5 (서열 1 및 서열 2)에 관한 것이다. 비피도박테륨 비피둠으로부터의 염색체 DNA를 PstI로 소화시킨 삽입물을 함유하는 플라스미드로 대장균 세포를 형질전환시켰다. 발색성 β-갈락토시다제 기질을 함유하는 플레이트상에서 β-갈락토시다제 활성을 보유한 클론을 선별했다. 양성 콜로니 중 하나는 대략 20 kb의 인서트인 pOLGA5 (서열 1)가 있는 플라스미드를 함유했다. DNA 서열결정을 통해 OLGA5 β-갈락토시다제의 추정되는 아미노산 서열 (서열 2)이 현재 공지된 β-갈락토시다제 길이의 대략 2배이며, 공지된 β-갈락토시다제와의 서열 상동성 정도가 놀라울 만큼 낮다는 것을 밝혀냈다. 대장균 내 재조합 OLGA5의 발현을 통해 상기 효소가 락토스 가수분해 활성 뿐 아니라 트랜스갈락토실화 활성까지 보유한다는 것을 밝혀냈다. OLGA5 효소의 C-말단부는 공지된 β-갈락토시다제와 상동성이 없었다. 이어서, OLGA5의 다양한 C-말단 결실 돌연변이체를 제작하고, 생성된 효소의 가수분해 활성 및 트랜스갈락토실화 활성에 대해 조사했다.
본 발명의 제2 측면은 OLGA5의 결실 돌연변이체, 예를 들어 OLGA347에 관한 것이다. 여러 C-말단 결실 돌연변이체 중에서, 578개 아미노산의 C-말단이 결실된 OLGA347이 비교적 저농도의 락토스와 인큐베이션시키는 경우일지라도 생산되는 올리고당류의 수준은 가장 두드러지게 증가된 것으로 밝혀졌다. 명백하게, 상기 효 소는 실제로 모든 갈락토스 분자를 갈락토스 또는 글루코스에 전달한다. 그러므로, OLGA5의 C-말단 종결부의 결실은 상기 효소를 가수분해성 OLGA5 β-갈락토시다제에서 트랜스갈락토실화 OLGA347-트랜스갈락토시다제로 전환시킨다. 천연 OLGA5 효소를 포함하는 다른 트랜스갈락토실화 β-갈락토시다제와는 달리, 상기 말단절단형 β-갈락토시다제 OLGA347은 시험된 모든 락토스 농도에서 갈락토스를 수용자 당 분자에 높은 빈도로 전달한다.
한 실시양태에서, OLGA5, OLGA342, OLGA345, OLGA347, OLGA344 또는 임의의 다른 OLGA5 변이체를 코딩하는 인서트를 보유하는 발현 벡터를 사용한다. 이러한 발현 벡터를 사용하여 비피도박테륨, 락토콕쿠스 (Lactococcus), 락토바실러스 (Lactobacillus), 스트렙토콕쿠스 (Streptococcus), 류코노스톡 (Leuconostoc), 에쉐리히아 (Escherichia), 바실러스 (Bacillus), 스트렙토마이세스 (Streptomyces), 사카로마이세스 (Saccharomyces), 클루이베로마이세스 (Kluyveromyces), 칸디다 (Candida), 토룰라 (Torula), 토룰롭시스 (Torulopsis) 및 아스페르길루스 (Aspergillus)를 포함하는 군에서 선택된 숙주 세포를 형질전환시킬 수 있다. 비피도박테륨 속의 세포는 비피도박테륨 브레브, 비피도박테륨 롱검, 비피도박테륨 인판티스 (Bifidobacterium infantis), 비피도박테륨 비피둠 및 락토콕쿠스 락티스 (Lactococcus lactis)로 구성된 군에서 선택한다. 이어서, 상기 세포를 적합한 배양 배지 중에서 OLGA5 또는 OLGA347 변이체 등의 발현을 허용하는 조건하에 배양한 후에, 생성된 효소를 배양물로부터 회수한다.
본 발명의 다른 실시양태에서, OLGA5 변이체는 상기 언급한 숙주 세포 중 임 의의 하나를 형질전환시킬 수 있는 발현 벡터의 일부이다. 이어서, 상기 세포를 적합한 배양 배지 중에서 OLGA5 변이체의 발현을 허용하는 조건하에 배양한 후에, 생성된 효소를 배양물로부터 회수한다. OLGA5 변이체는 임의의 무작위 돌연변이 또는 통상적인 분자 생물학 기술을 통해 제작한 임의의 돌연변이를 함유할 수 있다. 돌연변이되거나 야생형인 OLGA5 DNA 분자의 임의의 단편을 발현 벡터에 삽입할 수 있다. 상기 단편은 PCR (중합효소 연쇄 반응) 및(또는) 상기 서열에 존재하는 임의의 제한 부위를 이용하여 제작할 수 있다. OLGA5 변이체의 제작 방법은 당업자에게 공지되어 있다. 그러므로, 변이체 수득 방법은 본 발명에 중요하지 않다. 본원에 개시된 변이체는 상기 서열 중에 존재하는 제한 부위를 이용하여 서브클로닝함으로써 수득된다.
본 발명의 다른 측면은 요구르트, 치즈, 발효시킨 유제품, 식이보조제 및 건강보조 (probiotic) 식품으로 구성된 군에서 선택된 제품의 생산을 위한, 상기 언급한 세포 유형 중 1종 이상의 용도에 관한 것이다. 이러한 측면에서, 비피도박테륨의 성장 향상이라는 기술적 효과를 이용하여 산업적 제품의 품질을 개선시킨다. 갈락토-올리고당류의 첨가는 건강 촉진 비피도박테륨의 성장을 향상시킨다. 그러므로, OLGA347에 의해 생산된 갈락토-올리고당류는 올리고당류 생산을 위해 천연 β-갈락토시다제를 사용하는 경우에 비해 훨씬 저렴하고 수득 방법이 쉽다.
본 발명의 다른 측면은 올리고당류의 생산을 위한, OLGA5, OLGA342, OLGA345, OLGA347, OLGA344 또는 임의의 다른 OLGA5 변이체의 용도 또는 상기 언급한 세포 유형 중 1종 이상의 용도에 관한 것이다. 올리고당류로는 프룩토올리고당 류, 갈락토-올리고당류, 이소말토-올리고당류, 말토-올리고당류, 락토-수크로스 및 크실로-올리고당류 등이 있으나, 이에 제한되지 않는다.
본 발명의 한 실시양태에서, 이당류 기질, 예를 들어 락툴로스, 트레할로스, 람노스, 말토스, 수크로스, 락토스 또는 셀로비오스를 포함하는 배지 중에서 OLGA5 변이체를 발현하는 세포를 인큐베이션시켜 올리고당류를 생산한다. 인큐베이션은 올리고당류가 생산되는 조건하에 수행한다. 상기 세포는 요구르트, 치즈, 발효시킨 우유 제품, 식이보조제 및 건강보조 식품으로 구성된 군에서 선택된 제품의 일부일 수 있다. 별법으로, 올리고당류를 회수한 후에 이를 제조 전 또는 제조 후의 관심 제품에 첨가할 수 있다. 올리고당류의 첨가는 비피도박테륨 단독의 성장을 향상시키거나, 또는 혼합 배양물 중의 비피도박테륨의 성장을 향상시킨다.
다른 실시양태에서, 이당류 기질, 예를 들어 락툴로스, 트레할로스, 람노스, 말토스, 수크로스, 락토스 또는 셀로비오스를 포함하는 배지 중에서 OLGA5 변이체를 인큐베이션시켜 올리고당류를 생산한다. 인큐베이션은 올리고당류가 생산되는 조건하에 수행한다. OLGA5 변이체 및 락토스를 포함하는 배지는 요구르트, 치즈, 발효시킨 우유 제품, 식이보조제 및 건강보조 식품으로 구성된 군에서 선택된 제품의 일부일 수 있다. 별법으로, 올리고당류를 회수한 후에 이를 제조 전 또는 제조 후의 관심 제품에 첨가할 수 있다. 올리고당류의 첨가는 비피도박테륨 단독의 성장을 향상시키거나, 또는 혼합 배양물 중의 비피도박테륨의 성장을 향상시킨다.
정의
"β-갈락토시다제 또는 이의 단편": β-갈락토시다제는 락토스를 단당류인 D-글루코스 및 D-갈락토스로 가수분해할 수 있는 효소로서 정의된다. β-갈락토시다제의 단편은 상기 단백질의 5 내지 98%, 바람직하게는 40 내지 95% 및 가장 바람직하게는 55 내지 75%를 포함하며, 상기 결실은 C-말단 종결부가 결실된 것이 바람직하다.
"숙주 세포"는 잔균, 효모 및 원핵생물로 구성된 군에서 선택된다. 상기 미생물은 더욱 바람직하게는 원핵생물이고, 가장 바람직하게는 비피도박테륨 속 또는 대장균 종의 박테리아이다.
"올리고당류"란, 3개 이상의 당 분자로 구성된 올리고당류를 의미한다. 올리고당류의 예로는 갈락토-올리고당류 등이 있으나, 이에 제한되지 않는다. 올리고당류의 당 잔기 사이의 연결 방식은 1-4 및 1-6 결합을 포함하나, 이에 제한되지 않는다.
β-갈락토시다제와 락토스의 인큐베이션은 0.5 내지 60% 락토스, 바람직하게는 2 내지 30% 락토스 및 가장 바람직하게는 2 내지 15% 락토스의 존재하에서 수행한다.
β-갈락토시다제와 락토스를 인큐베이션시키는 조건은 5 내지 75℃, 바람직하게는 15 내지 45℃, 가장 바람직하게는 37℃의 온도에서 인큐베이션을 수행하는 것으로 정의된다. 인큐베이션에 필요한 시간은 1 내지 50시간, 바람직하게는 5 내지 40시간, 가장 바람직하게는 15 내지 25시간이다.
"식품"은 음식물, 음료수, 정제 및 분말 등의 섭취에 의도되는 제품을 포함한다.
실시예 1: 비피도박테륨 비피둠으로부터 트랜스갈락토실화 β- 갈락토시다제의 단리 및 특성화
비피도박테륨 비피둠 DSM 20215로부터의 염색체 DNA를 PstI로 소화시키고, 이를 표준 방법을 사용하여 pKS 플라스미드 (스트라타젠 (Stratagene) 제품)에 라이게이션시켰다. 상기 라이게이션 혼합물로 LacZ 및 β-갈락토시다제가 결핍된 대장균 균주 MT102를 형질전환시켰다. 발색성 β-갈락토시다제 기질인 X-gal을 함유하는 플레이트상에서 β-갈락토시다제를 생산하는 클론을 청색 콜로니로서 확인하였다.
청색 콜로니 중 하나는 대략 20 kb의 인서트인 pOLGA5가 있는 플라스미드를 함유했다. 상기 인서트를 추가로 서브클로닝하고 부분적으로 서열결정하였으며, 추정되는 β-갈락토시다제 (OLGA5 β-갈락토시다제)를 코딩하는 오픈 리딩 프레임을 확인하였다 (도 1). BLAST 검색을 통해, OLGA5 β-갈락토시다는 스트렙토마이세스 코엘리콜러 (Streptomyces coelicolor) β-갈락토시다제 (AL133171) 및 써모언에어로박터 에탄올리쿠스 (Thermoanaerobacter ethanolicus) (YO8557)와 가장 높은 상동성을 나타내어, 각각 38% 및 30% 동일성을 나타내는 것으로 밝혀졌다. 도 3은 OLGA5 및 관련된 아미노산 서열의 "동일성 트리 (tree)"를 나타낸다.
OLGA5 β-갈락토시다제 아미노산 서열에 관한 상세한 분석을 통해, 상기 효소가 이의 N-말단에 추정되는 신호 서열을 함유하고 오픈 리딩 프레임이 현재 공지된 임의의 β-갈락토시다제 크기의 대략 2배인 185 kDa의 폴리펩티드를 코딩한다는 것이 밝혀졌다. 대장균에서 생성된 재조합 OLGA5 효소를 정제하여 N-말단 아미노산을 서열결정한 결과, 대장균에서 발현되는 동안 신호 서열이 절단된다는 것을 확인하였다. SDS-PAGE를 통해, OLGA5 폴리펩티드의 분자량을 확인하였다.
pOLGA5를 함유하는 재조합 대장균 MT102의 세포 추출물을 제조하여 트랜스갈락토실화 활성에 대해 분석하였다. 도 4는 OLGA5가 락토스 가수분해 활성 뿐 아니라 트랜스갈락토실화 활성까지 보유한다는 것을 나타낸다.
실시예 2: 높은 트랜스갈락토실라제 활성을 보유한 말단절단형 OLGA5 β- 갈락토시 다제의 제조
OLGA5 중 다른 β-갈락토시다제와 상동성이 있는 영역은 상기 단백질의 N-말단 종결부에 위치한다. C-말단 절반은 임의의 공지된 β-갈락토시다제와 상동성이 없는 것으로 나타났다. 그러나, C-말단부에서 시알리다제-유사 갈락토스-결합 도메인이 관찰되었다. 말단절단형 결실 돌연변이체를 제조하여 OLGA5 β-갈락토시다제의 이러한 C-말단부의 역할을 조사하였다. 생성된 재조합 β-갈락토시다제의 가수분해 활성 및 트랜스갈락토실화 활성을 분석했다. 도 5는 OLGA5 효소의 거의 1/3을 절단하여도 여전히 가수분해 활성을 보유할 수 있음을 나타낸다.
트랜스갈락토실화 활성의 분석시에, 플라스미드 pOLGA5, pOLGA342 및 pOLGA345를 함유하는 대장균으로부터의 추출물을 사용하여 유사한 결과를 얻었다. 그러나, pOLGA347을 보유하는 세포 추출물에서는, 생산된 올리고당류의 수준은 증가되었으나 갈락토스는 전혀 생산되지 않은 것으로 나타났다. 도 5에 나타난 바와 같이, 말단절단형 OLGA347 β-갈락토시다제를 함유하는 추출물은 락토스를 가수분 해하지는 않았으나, 상기 효소는 갈락토스를 물의 히드록실기에 전달하는 대신 실제로 모든 갈락토스 분자를 갈락토스 또는 글루코스 (또는 글리세롤; TLC상에서 글루코스보다 다소 느리게 이동하는 스팟은 NMR에 의해 갈락토-글리세롤임이 밝혀졌음 - 데이타는 나타내지 않음)에 전달했다. 결론적으로, OLGA347은 진정한 "트랜스갈락토실라제"이다.
실시예
3:
OLGA347
의
트랜스갈락토실화
활성의 특성화
다음의 두가지 방법을 사용하여 OLGA347 β-갈락토시다제의 트랜스갈락토실화 활성을 정량하였다: 방사성 표지된 락토스를 함유하는 반응 혼합물의 TLC 분석 및 표지되지 않은 락토스를 효소적 전환시킨 후의 HPLC 분석.
글루코스의 C-1 위치에 14C 표지를 함유하는 락토스를 사용하여 방사성 실험을 수행했다. 표지는 상기 이당류의 글루코스 부분에 존재했기 때문에, 글루코스를 함유하는 반응 생성물만이 검출되었다. 도 7은 15% 락토스 및 다양한 양의 OLGA347 효소를 사용한 트랜스갈락토실화 실험의 결과를 나타낸다. TLC을 통해 반응 혼합물을 분리한 후, 상기 플레이트를 스캐닝하여 방사성 스팟을 포스포화상기에서 정량했다. 낮은 효소 농도 (추출물 0 내지 0.2 ㎕ 사이)에서 글루코스 및 올리고당류 수준은 거의 동일했으며, 이는 트랜스갈락토실화 반응에서 모든 글루코스 분자가 기질로서 활용되었음을 지시했다. 가수분해되어 "유리"된 글루코스는 효소가 고농도인 경우에만 나타났다.
표지되지 않은 락토스를 사용한 실험에서, 상이한 기질 및 효소 농도를 시험 하였다. 도 8은 OLGA347 효소의 농도를 변화시키면서 수행한 효소 반응에서 기질로서 10%, 20% 및 40% 락토스를 사용한 실험을 나타낸다. 반응 혼합물을 HPLC로 붙석하고, 락토스, 글루코스, 갈락토스 및 갈락토-올리고당류의 농도를 계산했다. 도 8은 효소 농도가 증가함에 따라 락토스 농도가 감소하고 글루코스 농도가 증가하지만, 실제로는 "유리" 갈락토스는 생산되지 않았음을 보여주며, 이는 락토스 중의 거의 모든 갈락토스 분자가 다른 당으로 전달되었음을 지시한다. 저농도의 효소를 사용한 반응에서 측정한 탄수화물의 농도 계산은 글루코스와 갈락토스의 비율이 대략 0.1임을 지시하는데, 이는 모든 락토스 분자가 유리 갈락토스 및 글루코스로 가수분해되며, 트랜스갈락토실화에는 9개의 락토스 분자가 사용된다는 것을 암시한다. 도 8에 나타난 바와 같이, 트랜스갈락토실화 반응은 10% 내지 40% 락토스 범위의 락토스 농도와는 무관하다. 10%, 20% 또는 40% 락토스를 기질로서 사용한 트랜스갈락토실화 반응에서 생산된 갈락토-올리고당류의 최대 수율은 각각 39%, 44% 및 37%였다 (첨가된 락토스 1 mg 당 생산된 올리고당류 (mg)).
참고문헌
도 1: OLGA5 서열. 비피도박테륨 비피둠의 OLGA5 β-갈락토시다제의 DNA 및 단백질 서열. 신호 서열은 굵은체로 나타내었고, OLGA347에서 결실된 OLGA5 유전자의 일부는 이탤릭체로 나타내었다. 결실 생성에 사용된 BglII 부위는 밝게 표시했다.
도 2: β-갈락토시다제 활성 부위 영역들의 비교. 대장균의 촉매적 Glu461 잔기의 주변 영역들의 정렬 (밝게 표시한 부분). 상기 서열은 이들의 데이타베이스 수탁 번호를 통해 확인하였다. 6-포스포-β-갈락토시다제 서열은 (P)로 표시했다.
도 3: 도 1에서의 정렬을 계통별로 연결 (neighbour joining)한 분석으로서, 술폴로부스 (Sulfolubus) 서열은 외(外)집단으로 사용했다. 부트스트랩 분석 (n = 100) 결과는 80 초과의 수치로 관련있음을 나타낸다.
도 4: OLGA5 트랜스갈락토실라제 활성. 플라스미드에 OLGA5 유전자를 보유하는 대장균 세포의 전체 세포 용균물을 0.4 M 락토스와 37℃에서 20시간 동안 인큐베이션했다. 총 반응 부피 50 ㎕는 전체 세포 용균물을 지시된 양만큼 함유했다. 반응 샘플을 실리카겔 TLC 플레이트상에서 분석했다. 상기 플레이트에 오르시놀 (Orcinol) 시약을 분무하여 당을 가시화했다.
도 5: OLGA5 β-갈락토시다제의 C-말단 결실. 1752개 아미노산의 오픈 리딩 프레임은 OLGA5 β-갈락토시다제를 코딩하며, 출발하는 32개 아미노산이 신호 펩티드 (백색 박스)를 나타내는 것으로 여겨진다. OLGA5의 결실 돌연변이체는 지시된 제한 부위를 사용하여 제작하였다. 밤새 성장시킨 박테리아 배양물로부터 제조한 용균물을 사용하여 β-갈락토시다제 활성을 측정하고, 상대적인 결과를 각 구조물의 오른쪽에 나타냈다. 사용한 제한 효소 부호: BglII (B), EcoRI (E), EcoRV (V), HindIII (H), KpnI (K), NruI (N), PstI (P).
도 6: 트랜스갈락토실라제 활성의 TLC 분석. 시험한 2가지 결실 돌연변이체인 OLGA347 및 OLGA345의 전체 세포 용균물을 지시된 양으로 사용하여 총 부피 50 ㎕로 0.4 M 락토스와 반응시켰다. 반응물을 37℃에서 20시간 동안 인큐베이션했다. 샘플을 실리카겔 TLC 플레이트상에서 분석했다. 상기 플레이트에 오르시놀 시약을 분무하여 당을 가시화했다.
도 7: OLGA347에 의해 생산된 올리고당류. OLGA347 전체 세포 용균물을 지시된 양으로 사용하여 총 부피 50 ㎕로 15% 락토스와 37℃에서 21시간 동안 인큐베이션했다. 글루코스 C-1 위치에 14C로 표지한 방사성 락토스를 사용했다. 샘플들을 TLC 플레이트상에서 분리하고 포스포화상기 (phosphoimager)를 사용하여 정량하였다. A: 락토스, 글루코스 및 갈락토-올리고당류 (GOS) 스팟으로부터의 14C 신호 측정에 사용한 화상; B: 백그라운드 (background) (빈 레인)를 감하여 측정한 14C-신호.
도 8: OLGA347 효소 반응 생성물의 HPLC 측정. OLGA347 전체 세포 용균물을 지시된 양으로 사용하여 10%, 20% 및 40% 락토스와의 반응을 수행했다. 총 부피를 200 ㎕로 사용하였고, 반응물을 37℃에서 20시간 동안 인큐베이션했다. 희석 한 샘플을 HPLC 분석하였고, 표준 곡선을 사용하여 관찰된 피크 영역을 농도 (mg/mL)로 전환시켰다. A: OLGA347이 10% 락토스와 반응한 후에 수득된 당 (mg/mL); B: OLGA347이 20% 락토스와 반응한 후에 수득된 당 (mg/mL); C: OLGA347이 40% 락토스와 반응한 후에 수득된 당 (mg/mL); D: 상기 10% 반응 결과의 플롯. 갈락토-올리고당류의 생산량을 글루코스 또는 갈락토스로서 회수되지 않은 락토스의 양으로서 계산했다 ("GOS").
<110> ARLA FOODS AMBA
<120> Beta-Galactosidase Isolated from a Bifidobacterium
<130> PC025441
<150> US 60/207,154
<151> 2000-05-26
<160> 2
<170> KopatentIn 1.71
<210> 1
<211> 5509
<212> DNA
<213> Bifidobacterium bifidum
<220>
<221> CDS
<222> (212)..(5467)
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630 635 640 645
ttg gcc gtc gcc gaa gaa ctc gta ctt cgg cat cgt cga cac cgc agg 2194
Leu Ala Val Ala Glu Glu Leu Val Leu Arg His Arg Arg His Arg Arg
650 655 660
ctt ccc gaa gac acc tat tac ttc tat cag agc cag tgg aac gac gac 2242
Leu Pro Glu Asp Thr Tyr Tyr Phe Tyr Gln Ser Gln Trp Asn Asp Asp
665 670 675
gtg cac acg ctg cac atc ctc ccc gca tgg aac gag aac gtc gtc gcc 2290
Val His Thr Leu His Ile Leu Pro Ala Trp Asn Glu Asn Val Val Ala
680 685 690
aag ggc tcc ggc aac aac gtg ccg gtc gtc gtc tac acc gac gcg gcc 2338
Lys Gly Ser Gly Asn Asn Val Pro Val Val Val Tyr Thr Asp Ala Ala
695 700 705
aag gtc aag ctg tac ttc aca ccg aag ggc agt acc gaa aag cga ctg 2386
Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser Thr Glu Lys Arg Leu
710 715 720 725
atc gga gag aag tcc ttc acc aag aag acc acc gcg gcc gga tac acc 2434
Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr Ala Ala Gly Tyr Thr
730 735 740
tat cag gtc tac gag ggc tcc gac aag gac tcc acc gcc cac aag aac 2482
Tyr Gln Val Tyr Glu Gly Ser Asp Lys Asp Ser Thr Ala His Lys Asn
745 750 755
atg tac ctg acc tgg aac gtg ccg tgg gcc gag ggc acc atc tcc gcc 2530
Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu Gly Thr Ile Ser Ala
760 765 770
gaa gca tac gac gag aac aac agg ctg atc ccc gag ggg tcc acc gag 2578
Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro Glu Gly Ser Thr Glu
775 780 785
ggc aac gcg tcg gtg acc acc acc ggc aag gcc gcg aag ctt aaa gcc 2626
Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala Ala Lys Leu Lys Ala
790 795 800 805
gat gcc gac cgc aag acg atc acc gcg gac ggc aag gac ctg tcg tac 2674
Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly Lys Asp Leu Ser Tyr
810 815 820
atc gag gtc gac gtg acc gac gcc aac ggc cat atc gtc ccc gat gcc 2722
Ile Glu Val Asp Val Thr Asp Ala Asn Gly His Ile Val Pro Asp Ala
825 830 835
gcc aac cgc gtc acc ttc gac gtc aag ggc gcc ggc aaa ctg gtc ggc 2770
Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala Gly Lys Leu Val Gly
840 845 850
gtc gac aac ggc agc tcg ccg gat cac gac tcc tat cag gcc gac aac 2818
Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser Tyr Gln Ala Asp Asn
855 860 865
cgc aag gcg ttc agc ggc aag gtg ctc gcc atc gtc cag tcc acc aag 2866
Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile Val Gln Ser Thr Lys
870 875 880 885
gag gcg ggc gag atc acc gtc acc gcc aag gcc gac ggt ctg caa tca 2914
Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala Asp Gly Leu Gln Ser
890 895 900
tcc aca gtg aag atc gcc acc acc gcc gtc ccc ggc acc agc acc gag 2962
Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro Gly Thr Ser Thr Glu
905 910 915
aag acg gtc cgc agc ttc tac tac tcg cgc aac tac tac gtc aag acc 3010
Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn Tyr Tyr Val Lys Thr
920 925 930
ggc aac aag ccg att ctg ccg agt gat gtc gag gtg cgc tac tcc gac 3058
Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu Val Arg Tyr Ser Asp
935 940 945
ggc acg tcg gac cgt cag aac gtc aca tgg gac gca gtc agc gac gac 3106
Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp Ala Val Ser Asp Asp
950 955 960 965
cag atc gcc aag gcc ggt tcg ttc agc gtg gcc ggc acg gtc gcc ggg 3154
Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala Gly Thr Val Ala Gly
970 975 980
cag aag atc tcc gtg cgc gtg acg atg atc gac gag atc ggt gcg ctg 3202
Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp Glu Ile Gly Ala Leu
985 990 995
ctc aac tat tcg gcc agc aca ccg gtc ggc acg ccc gcc gtg ctg cct 3250
Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr Pro Ala Val Leu Pro
1000 1005 1010
ggc tcg cgt ccg gcc gtg ctg ccc gac ggc acc gtg acc agc gcg aac 3298
Gly Ser Arg Pro Ala Val Leu Pro Asp Gly Thr Val Thr Ser Ala Asn
1015 1020 1025
ttc gcc gtc cac tgg acc aag ccc gcc gac acc gtg tac aac acg gcc 3346
Phe Ala Val His Trp Thr Lys Pro Ala Asp Thr Val Tyr Asn Thr Ala
1030 1035 1040 1045
ggc acc gtc aag gtc ccc ggc acc gcc acc gtc ttc ggc aag gag ttc 3394
Gly Thr Val Lys Val Pro Gly Thr Ala Thr Val Phe Gly Lys Glu Phe
1050 1055 1060
aag gtc acc gcg acg att cgc gtg cag cgg tcg cag gtc acc atc ggc 3442
Lys Val Thr Ala Thr Ile Arg Val Gln Arg Ser Gln Val Thr Ile Gly
1065 1070 1075
agc agc gtc tcc ggc aat gcg ctg cgc ctg act cag aac atc ccc gcc 3490
Ser Ser Val Ser Gly Asn Ala Leu Arg Leu Thr Gln Asn Ile Pro Ala
1080 1085 1090
gac aag cag tcc gac acg ctg gac gcc atc aag gac ggc tcc acg acc 3538
Asp Lys Gln Ser Asp Thr Leu Asp Ala Ile Lys Asp Gly Ser Thr Thr
1095 1100 1105
gtc gac gcc aat acc ggc ggc ggc gcg aac ccg tca gca tgg acc aac 3586
Val Asp Ala Asn Thr Gly Gly Gly Ala Asn Pro Ser Ala Trp Thr Asn
1110 1115 1120 1125
tgg gcg tac tcg aag gcc ggc cac aac acc gcc gag atc acc ttc gag 3634
Trp Ala Tyr Ser Lys Ala Gly His Asn Thr Ala Glu Ile Thr Phe Glu
1130 1135 1140
tac gcg acc gag cag cag ctc ggc cag att gtc atg tac ttc ttc cgc 3682
Tyr Ala Thr Glu Gln Gln Leu Gly Gln Ile Val Met Tyr Phe Phe Arg
1145 1150 1155
gac agc aac gcg gtg agg ttc ccc gac gcc ggc aag acg aag atc cag 3730
Asp Ser Asn Ala Val Arg Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln
1160 1165 1170
atc tcc gcg gac ggc aag aac tgg acg gat ctc gct gcc acg gag acc 3778
Ile Ser Ala Asp Gly Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu Thr
1175 1180 1185
atc gcg gcc cag gag tcg tcc gac cga gtc aag ccg tac acc tat gac 3826
Ile Ala Ala Gln Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr Tyr Asp
1190 1195 1200 1205
ttc gct ccg gtg gga gcc acg ttc gtc aag gtc acg gtc acc aac gcc 3874
Phe Ala Pro Val Gly Ala Thr Phe Val Lys Val Thr Val Thr Asn Ala
1210 1215 1220
gac acc aca acc ccc agc ggc gtg gtc tgc gcc ggc ctg acc gag atc 3922
Asp Thr Thr Thr Pro Ser Gly Val Val Cys Ala Gly Leu Thr Glu Ile
1225 1230 1235
gag ctg aag acc gcg acc agc aag ttc gtc acg aac acg tcc gcc gcg 3970
Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr Asn Thr Ser Ala Ala
1240 1245 1250
ctc tcg tcg ctg aca gtg aac ggc acg aag gtc tcc gac tcc gtg ctc 4018
Leu Ser Ser Leu Thr Val Asn Gly Thr Lys Val Ser Asp Ser Val Leu
1255 1260 1265
gcc gcc ggc tcc tac aac acg ccc gcg atc atc gcg gac gtc aaa gcc 4066
Ala Ala Gly Ser Tyr Asn Thr Pro Ala Ile Ile Ala Asp Val Lys Ala
1270 1275 1280 1285
gag ggc gaa ggc aac gcc agc gtc acc gtg ctg ccc gcg cac gac aac 4114
Glu Gly Glu Gly Asn Ala Ser Val Thr Val Leu Pro Ala His Asp Asn
1290 1295 1300
gtg atc cgc gtg atc acc gag tcc gag gac cac gtc acg cgc aag acc 4162
Val Ile Arg Val Ile Thr Glu Ser Glu Asp His Val Thr Arg Lys Thr
1305 1310 1315
ttc acc atc aac ctg ggc acg gag cag gaa ttc ccc gca gac tcc gat 4210
Phe Thr Ile Asn Leu Gly Thr Glu Gln Glu Phe Pro Ala Asp Ser Asp
1320 1325 1330
gaa cgc gac tac ccg gcc gcc gac atg acg gtc acc gtg ggc agc gaa 4258
Glu Arg Asp Tyr Pro Ala Ala Asp Met Thr Val Thr Val Gly Ser Glu
1335 1340 1345
cag acg tcc ggc acc gcg acc gaa ggc ccg aag aaa ttc gcg gtc gac 4306
Gln Thr Ser Gly Thr Ala Thr Glu Gly Pro Lys Lys Phe Ala Val Asp
1350 1355 1360 1365
ggc aac acc agc acg tac tgg cat tcc aac tgg acg ccc acc acc gtg 4354
Gly Asn Thr Ser Thr Tyr Trp His Ser Asn Trp Thr Pro Thr Thr Val
1370 1375 1380
aac gac ctg tgg atc gcc ttc gag ctc cag aaa ccc acc aag ctc gac 4402
Asn Asp Leu Trp Ile Ala Phe Glu Leu Gln Lys Pro Thr Lys Leu Asp
1385 1390 1395
gcg ctg cgc tac ctg ccg cgc ccc gcg ggc agc aag aac ggc tcc gtc 4450
Ala Leu Arg Tyr Leu Pro Arg Pro Ala Gly Ser Lys Asn Gly Ser Val
1400 1405 1410
acc gaa tac aag gtt cag gtc agc gat gac ggc acc aac tgg acc gac 4498
Thr Glu Tyr Lys Val Gln Val Ser Asp Asp Gly Thr Asn Trp Thr Asp
1415 1420 1425
gcg ggc tcc ggc aca tgg acc acc gat tac ggc tgg aag ctc gcc gag 4546
Ala Gly Ser Gly Thr Trp Thr Thr Asp Tyr Gly Trp Lys Leu Ala Glu
1430 1435 1440 1445
ttc aat cag ccg gtg acc acc aag cac gtg cgg ctc aag gcc gtc cac 4594
Phe Asn Gln Pro Val Thr Thr Lys His Val Arg Leu Lys Ala Val His
1450 1455 1460
acc tat gcg gat tcc ggc aac gac aag ttc atg tcc gcc tcc gaa atc 4642
Thr Tyr Ala Asp Ser Gly Asn Asp Lys Phe Met Ser Ala Ser Glu Ile
1465 1470 1475
cgc ctg cgc aag gcc gtc gac acc acc gac atc agc ggc gcg acc gtg 4690
Arg Leu Arg Lys Ala Val Asp Thr Thr Asp Ile Ser Gly Ala Thr Val
1480 1485 1490
acc gtg ccc gcc aag ctg acc gtc gac cgg gtg gac gcc gac cat ccc 4738
Thr Val Pro Ala Lys Leu Thr Val Asp Arg Val Asp Ala Asp His Pro
1495 1500 1505
gcc acc ttc gcc acg aag gac gtg acg gtg acg ttg ggc gac gcc acg 4786
Ala Thr Phe Ala Thr Lys Asp Val Thr Val Thr Leu Gly Asp Ala Thr
1510 1515 1520 1525
ctg cgc tac ggc gtg gac tac ctg ctc gac tac gcg ggc aac acc gcc 4834
Leu Arg Tyr Gly Val Asp Tyr Leu Leu Asp Tyr Ala Gly Asn Thr Ala
1530 1535 1540
gtc ggc aag gcc acg gtg acc gtg cgc ggc atc gac aag tac tcc ggc 4882
Val Gly Lys Ala Thr Val Thr Val Arg Gly Ile Asp Lys Tyr Ser Gly
1545 1550 1555
acc gtc gcc aag acg ttc acc atc gaa ctg aag aac gcc ccg gcg ccg 4930
Thr Val Ala Lys Thr Phe Thr Ile Glu Leu Lys Asn Ala Pro Ala Pro
1560 1565 1570
gaa ccg acg ctg acc tcg gtg agc gtc aag acc aag cct tcc aag ctg 4978
Glu Pro Thr Leu Thr Ser Val Ser Val Lys Thr Lys Pro Ser Lys Leu
1575 1580 1585
acc tat gtg gtc ggc gac gcg ttc gac ccg gca gga ctg gtg ctg cag 5026
Thr Tyr Val Val Gly Asp Ala Phe Asp Pro Ala Gly Leu Val Leu Gln
1590 1595 1600 1605
cac gac aga cag gcc gat cgc ccc cca cag cca ctt gtt gga gaa cag 5074
His Asp Arg Gln Ala Asp Arg Pro Pro Gln Pro Leu Val Gly Glu Gln
1610 1615 1620
gcc gac gaa cgc gga ctg acg tgc gga acg cga tgc gat cgc gtt gaa 5122
Ala Asp Glu Arg Gly Leu Thr Cys Gly Thr Arg Cys Asp Arg Val Glu
1625 1630 1635
cag ctg cgc aaa cac gag aat cgt gaa gcc cat cgt acg ggc ctc gat 5170
Gln Leu Arg Lys His Glu Asn Arg Glu Ala His Arg Thr Gly Leu Asp
1640 1645 1650
cat ctg gaa ttc gtg ggt gcc gcc gat gga gcg gtc ggt gaa cag gcc 5218
His Leu Glu Phe Val Gly Ala Ala Asp Gly Ala Val Gly Glu Gln Ala
1655 1660 1665
acc ttc aag gtg cat gtc cat gcc gat caa ggt gac ggc cgc cat gat 5266
Thr Phe Lys Val His Val His Ala Asp Gln Gly Asp Gly Arg His Asp
1670 1675 1680 1685
gat gcc gat gaa cgc gat atc gat cca cat gtc cct gtc gat cac gcg 5314
Asp Ala Asp Glu Arg Asp Ile Asp Pro His Val Pro Val Asp His Ala
1690 1695 1700
gtc ggt gag ctt gcg cgg gct gcg tgc cat cac gtc atc ggt ctg cgg 5362
Val Gly Glu Leu Ala Arg Ala Ala Cys His His Val Ile Gly Leu Arg
1705 1710 1715
gtc gac acc cat cgc ctc aag gca tcc ggc ttc cag atc ccc gcc gac 5410
Val Asp Thr His Arg Leu Lys Ala Ser Gly Phe Gln Ile Pro Ala Asp
1720 1725 1730
gac atg gcc gag atc gac cgc atc acc ggc ttc cac cgc ttc gag cgc 5458
Asp Met Ala Glu Ile Asp Arg Ile Thr Gly Phe His Arg Phe Glu Arg
1735 1740 1745
cac gtc ggc tga cgtgattggg cttccccgct gtctggtgcc ggctcgcga 5509
His Val Gly
1750
<210> 2
<211> 1752
<212> PRT
<213> Bifidobacterium bifidum
<400> 2
Met Ala Val Arg Arg Leu Gly Gly Arg Ile Val Ala Phe Ala Ala Thr
1 5 10 15
Val Ala Leu Ser Ile Pro Leu Gly Leu Leu Thr Asn Ser Ala Trp Ala
20 25 30
Val Glu Asp Ala Thr Arg Ser Asp Ser Thr Thr Gln Met Ser Ser Thr
35 40 45
Pro Glu Val Val Tyr Ser Ser Ala Val Asp Ser Lys Gln Asn Arg Thr
50 55 60
Ser Asp Phe Asp Ala Asn Trp Lys Phe Met Leu Ser Asp Ser Val Gln
65 70 75 80
Ala Gln Asp Pro Ala Phe Asp Asp Ser Ala Trp Gln Gln Val Asp Leu
85 90 95
Pro His Asp Tyr Ser Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu Ala
100 105 110
Glu Ser Ala Tyr Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser Phe
115 120 125
Thr Ile Asp Arg Asp Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe Asp
130 135 140
Gly Val Tyr Met Asn Ala Thr Val Trp Phe Asn Gly Val Lys Leu Gly
145 150 155 160
Thr His Pro Tyr Gly Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly Asn
165 170 175
Ala Lys Phe Gly Gly Glu Asn Thr Ile Val Val Lys Val Glu Asn Arg
180 185 190
Leu Pro Ser Ser Arg Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val
195 200 205
Thr Leu Thr Val Thr Asp Gly Val His Val Gly Asn Asn Gly Val Ala
210 215 220
Ile Lys Thr Pro Ser Leu Ala Thr Gln Asn Gly Gly Asp Val Thr Met
225 230 235 240
Asn Leu Thr Thr Lys Val Ala Asn Asp Thr Glu Ala Ala Ala Asn Ile
245 250 255
Thr Leu Lys Gln Thr Val Phe Pro Lys Gly Gly Lys Thr Asp Ala Ala
260 265 270
Ile Gly Thr Val Thr Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala Ser
275 280 285
Ala Asp Val Thr Ser Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp Ser
290 295 300
Ile Lys Asn Pro Asn Leu Tyr Thr Val Arg Thr Glu Val Leu Asn Gly
305 310 315 320
Gly Lys Val Leu Asp Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp Thr
325 330 335
Gly Phe Asp Ala Thr Ser Gly Phe Ser Leu Asn Gly Glu Lys Val Lys
340 345 350
Leu Lys Gly Val Ser Met His His Asp Gln Gly Ser Leu Gly Ala Val
355 360 365
Ala Asn Arg Arg Ala Ile Glu Arg Gln Val Glu Ile Leu Gln Lys Met
370 375 380
Gly Val Asn Ser Ile Arg Thr Thr His Asn Pro Ala Ala Lys Ala Leu
385 390 395 400
Ile Asp Val Cys Asn Glu Lys Gly Val Leu Val Val Glu Glu Val Phe
405 410 415
Asp Met Trp Asn Arg Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly Lys
420 425 430
Trp Phe Gly Gln Ala Ile Ala Gly Asp Asn Ala Val Leu Gly Gly Asp
435 440 445
Lys Asp Glu Thr Trp Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn Arg
450 455 460
Asp Arg Asn Ala Pro Ser Val Ile Met Trp Ser Leu Gly Asn Glu Met
465 470 475 480
Met Glu Gly Ile Ser Gly Ser Val Ser Gly Phe Pro Ala Thr Ser Ala
485 490 495
Lys Leu Val Ala Trp Thr Lys Ala Ala Asp Ser Thr Arg Pro Met Thr
500 505 510
Tyr Gly Asp Asn Lys Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr Met
515 520 525
Gly Asp Asn Leu Thr Ala Asn Gly Gly Val Val Gly Thr Asn Tyr Ser
530 535 540
Asp Gly Ala Asn Tyr Asp Lys Ile Arg Thr Thr His Pro Ser Trp Ala
545 550 555 560
Ile Tyr Gly Ser Glu Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile Tyr
565 570 575
Asn Arg Thr Thr Gly Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr Ser
580 585 590
Tyr Asp Asn Ser Ala Val Gly Trp Gly Ala Val Ala Ser Ser Ala Trp
595 600 605
Tyr Asp Val Val Gln Arg Asp Phe Val Ala Gly Thr Tyr Val Trp Thr
610 615 620
Gly Phe Asp Tyr Leu Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly Ser
625 630 635 640
Gly Ala Val Gly Ser Leu Ala Val Ala Glu Glu Leu Val Leu Arg His
645 650 655
Arg Arg His Arg Arg Leu Pro Glu Asp Thr Tyr Tyr Phe Tyr Gln Ser
660 665 670
Gln Trp Asn Asp Asp Val His Thr Leu His Ile Leu Pro Ala Trp Asn
675 680 685
Glu Asn Val Val Ala Lys Gly Ser Gly Asn Asn Val Pro Val Val Val
690 695 700
Tyr Thr Asp Ala Ala Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser
705 710 715 720
Thr Glu Lys Arg Leu Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr
725 730 735
Ala Ala Gly Tyr Thr Tyr Gln Val Tyr Glu Gly Ser Asp Lys Asp Ser
740 745 750
Thr Ala His Lys Asn Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu
755 760 765
Gly Thr Ile Ser Ala Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro
770 775 780
Glu Gly Ser Thr Glu Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala
785 790 795 800
Ala Lys Leu Lys Ala Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly
805 810 815
Lys Asp Leu Ser Tyr Ile Glu Val Asp Val Thr Asp Ala Asn Gly His
820 825 830
Ile Val Pro Asp Ala Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala
835 840 845
Gly Lys Leu Val Gly Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser
850 855 860
Tyr Gln Ala Asp Asn Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile
865 870 875 880
Val Gln Ser Thr Lys Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala
885 890 895
Asp Gly Leu Gln Ser Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro
900 905 910
Gly Thr Ser Thr Glu Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn
915 920 925
Tyr Tyr Val Lys Thr Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu
930 935 940
Val Arg Tyr Ser Asp Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp
945 950 955 960
Ala Val Ser Asp Asp Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala
965 970 975
Gly Thr Val Ala Gly Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp
980 985 990
Glu Ile Gly Ala Leu Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr
995 1000 1005
Pro Ala Val Leu Pro Gly Ser Arg Pro Ala Val Leu Pro Asp Gly Thr
1010 1015 1020
Val Thr Ser Ala Asn Phe Ala Val His Trp Thr Lys Pro Ala Asp Thr
1025 1030 1035 1040
Val Tyr Asn Thr Ala Gly Thr Val Lys Val Pro Gly Thr Ala Thr Val
1045 1050 1055
Phe Gly Lys Glu Phe Lys Val Thr Ala Thr Ile Arg Val Gln Arg Ser
1060 1065 1070
Gln Val Thr Ile Gly Ser Ser Val Ser Gly Asn Ala Leu Arg Leu Thr
1075 1080 1085
Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp Thr Leu Asp Ala Ile Lys
1090 1095 1100
Asp Gly Ser Thr Thr Val Asp Ala Asn Thr Gly Gly Gly Ala Asn Pro
1105 1110 1115 1120
Ser Ala Trp Thr Asn Trp Ala Tyr Ser Lys Ala Gly His Asn Thr Ala
1125 1130 1135
Glu Ile Thr Phe Glu Tyr Ala Thr Glu Gln Gln Leu Gly Gln Ile Val
1140 1145 1150
Met Tyr Phe Phe Arg Asp Ser Asn Ala Val Arg Phe Pro Asp Ala Gly
1155 1160 1165
Lys Thr Lys Ile Gln Ile Ser Ala Asp Gly Lys Asn Trp Thr Asp Leu
1170 1175 1180
Ala Ala Thr Glu Thr Ile Ala Ala Gln Glu Ser Ser Asp Arg Val Lys
1185 1190 1195 1200
Pro Tyr Thr Tyr Asp Phe Ala Pro Val Gly Ala Thr Phe Val Lys Val
1205 1210 1215
Thr Val Thr Asn Ala Asp Thr Thr Thr Pro Ser Gly Val Val Cys Ala
1220 1225 1230
Gly Leu Thr Glu Ile Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr
1235 1240 1245
Asn Thr Ser Ala Ala Leu Ser Ser Leu Thr Val Asn Gly Thr Lys Val
1250 1255 1260
Ser Asp Ser Val Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala Ile Ile
1265 1270 1275 1280
Ala Asp Val Lys Ala Glu Gly Glu Gly Asn Ala Ser Val Thr Val Leu
1285 1290 1295
Pro Ala His Asp Asn Val Ile Arg Val Ile Thr Glu Ser Glu Asp His
1300 1305 1310
Val Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly Thr Glu Gln Glu Phe
1315 1320 1325
Pro Ala Asp Ser Asp Glu Arg Asp Tyr Pro Ala Ala Asp Met Thr Val
1330 1335 1340
Thr Val Gly Ser Glu Gln Thr Ser Gly Thr Ala Thr Glu Gly Pro Lys
1345 1350 1355 1360
Lys Phe Ala Val Asp Gly Asn Thr Ser Thr Tyr Trp His Ser Asn Trp
1365 1370 1375
Thr Pro Thr Thr Val Asn Asp Leu Trp Ile Ala Phe Glu Leu Gln Lys
1380 1385 1390
Pro Thr Lys Leu Asp Ala Leu Arg Tyr Leu Pro Arg Pro Ala Gly Ser
1395 1400 1405
Lys Asn Gly Ser Val Thr Glu Tyr Lys Val Gln Val Ser Asp Asp Gly
1410 1415 1420
Thr Asn Trp Thr Asp Ala Gly Ser Gly Thr Trp Thr Thr Asp Tyr Gly
1425 1430 1435 1440
Trp Lys Leu Ala Glu Phe Asn Gln Pro Val Thr Thr Lys His Val Arg
1445 1450 1455
Leu Lys Ala Val His Thr Tyr Ala Asp Ser Gly Asn Asp Lys Phe Met
1460 1465 1470
Ser Ala Ser Glu Ile Arg Leu Arg Lys Ala Val Asp Thr Thr Asp Ile
1475 1480 1485
Ser Gly Ala Thr Val Thr Val Pro Ala Lys Leu Thr Val Asp Arg Val
1490 1495 1500
Asp Ala Asp His Pro Ala Thr Phe Ala Thr Lys Asp Val Thr Val Thr
1505 1510 1515 1520
Leu Gly Asp Ala Thr Leu Arg Tyr Gly Val Asp Tyr Leu Leu Asp Tyr
1525 1530 1535
Ala Gly Asn Thr Ala Val Gly Lys Ala Thr Val Thr Val Arg Gly Ile
1540 1545 1550
Asp Lys Tyr Ser Gly Thr Val Ala Lys Thr Phe Thr Ile Glu Leu Lys
1555 1560 1565
Asn Ala Pro Ala Pro Glu Pro Thr Leu Thr Ser Val Ser Val Lys Thr
1570 1575 1580
Lys Pro Ser Lys Leu Thr Tyr Val Val Gly Asp Ala Phe Asp Pro Ala
1585 1590 1595 1600
Gly Leu Val Leu Gln His Asp Arg Gln Ala Asp Arg Pro Pro Gln Pro
1605 1610 1615
Leu Val Gly Glu Gln Ala Asp Glu Arg Gly Leu Thr Cys Gly Thr Arg
1620 1625 1630
Cys Asp Arg Val Glu Gln Leu Arg Lys His Glu Asn Arg Glu Ala His
1635 1640 1645
Arg Thr Gly Leu Asp His Leu Glu Phe Val Gly Ala Ala Asp Gly Ala
1650 1655 1660
Val Gly Glu Gln Ala Thr Phe Lys Val His Val His Ala Asp Gln Gly
1665 1670 1675 1680
Asp Gly Arg His Asp Asp Ala Asp Glu Arg Asp Ile Asp Pro His Val
1685 1690 1695
Pro Val Asp His Ala Val Gly Glu Leu Ala Arg Ala Ala Cys His His
1700 1705 1710
Val Ile Gly Leu Arg Val Asp Thr His Arg Leu Lys Ala Ser Gly Phe
1715 1720 1725
Gln Ile Pro Ala Asp Asp Met Ala Glu Ile Asp Arg Ile Thr Gly Phe
1730 1735 1740
His Arg Phe Glu Arg His Val Gly
1745 1750
Claims (33)
- a) 서열 2의 아미노산 서열을 갖는 단백질을 코딩하는 DNA 서열,b) 엄격한 조건하에 (a)의 서열과 혼성화하는 DNA 서열, 또는c) (a) 또는 (b)의 서열과 동의성이 있는 (degenerative) DNA 서열.
- 제1항에 있어서, 서열 1의 서열인 DNA 서열 또는 이의 단편.
- 제2항에 있어서, 서열 1의 위치 212 내지 214의 ATG로 출발하여 위치 5468 내지 5470의 TGA에서 종결하는 서열을 포함하는 DNA 서열 또는 이의 임의의 단편.
- 제3항에 있어서, 서열 1의 위치 212 내지 214의 ATG로 출발하여 위치 3731 내지 3734의 ATCT에서 종결하는 서열을 포함하는 DNA 서열 또는 이의 임의의 단편.
- 제3항에 있어서, 서열 1의 위치 308 내지 310의 GTC로 출발하여 위치 5468 내지 5470의 TGA에서 종결하는 서열을 포함하는 DNA 서열 또는 이의 임의의 단편.
- 제3항에 있어서, 서열 1의 위치 308 내지 310의 GTC로 출발하여 위치 3731 내지 3734의 ATCT에서 종결하는 서열을 포함하는 DNA 서열 또는 이의 임의의 단편.
- 제1항 내지 제6항 중 어느 한 항에 있어서, 서열 2에 따른 아미노산 서열의 60% 미만, 바람직하게는 45% 미만, 더욱 바람직하게는 25% 미만이 변화되는 뉴클레오티드 치환, 부가 또는 결실을 포함하는 DNA 서열 또는 이의 단편.
- 제1항 내지 제5항 중 어느 한 항에 있어서, 보존적 아미노산 치환되는 뉴클레오티드 치환을 포함하는 DNA 서열.
- 제1항 내지 제8항 중 어느 한 항의 DNA 서열에 의해 코딩되는 효소.
- 서열 2에 따른 아미노산 서열 또는 이의 단편을 포함하는 효소.
- 서열 2에 정의된 바와 같은 서열을 갖는 β-갈락토시다제.
- 제10항에 있어서, 서열 2에 정의된 바와 같은 Met(1) 내지 Gly(1752)의 서열 또는 이의 단편을 갖는 효소.
- 제12항에 따른 성숙한 β-갈락토시다제.
- 제10항에 있어서, 서열 2에 정의된 바와 같은 Met(1) 내지 Ile(1174)의 서열 또는 이의 단편을 갖는 효소.
- 제14항에 따른 트랜스갈락토실화 효소.
- 제14항에 있어서, 서열 2에 정의된 바와 같은 Ala(33) 내지 Ile(1174)의 서열 또는 이의 단편을 갖는 효소.
- 제16항에 따른 성숙한 트랜스갈락토실화 효소.
- 제14항 내지 제17항 중 어느 한 항에 있어서,a) 37℃에서 100 g/L 락토스 용액 중에서 β-갈락토시다제 활성에 대한 트랜스갈락토실화 활성의 비가 1 : 1 이상임,b) 37℃에서 100 g/L 락토스 용액을 사용한 배치 (batch) 반응에서 25% 이상의 갈락토-올리고당류 생산을 촉매함,c) 37℃에서 100 g/L 락토스 용액을 사용한 배치 반응에서 갈락토-올리고당류 생산을 촉매하며 갈락토-올리고당류의 농도가 최고인 반응 시간에 유리 형태로 존재하는 락토스로부터 15% 미만의 갈락토스 생산을 촉매함의 특징 중 하나 이상을 보유하는 트랜스갈락토실화 효소.
- 제1항 내지 제8항 중 어느 한 항의 DNA 서열을 포함하는 재조합 벡터.
- 제19항에 있어서, 발현 벡터인 벡터.
- 제1항 내지 제8항 중 어느 한 항의 DNA 서열을 포함하는 숙주 세포.
- 제19항 또는 제20항의 벡터를 포함하는 숙주 세포.
- 제21항 또는 제22항에 있어서, 박테리아 세포, 효모 세포 또는 진균 세포인 세포.
- 제23항에 있어서, 비피도박테륨 (Bifidobacterium), 락토콕쿠스 (Lactococcus), 락토바실러스 (Lactobacillus), 스트렙토콕쿠스 (Streptococcus), 류코노스톡 (Leuconostoc), 에쉐리히아 (Escherichia), 바실러스 (Bacillus), 스트렙토마이세스 (Streptomyces), 사카로마이세스 (Saccharomyces), 클루이베로마이세스 (Kluyveromyces), 칸디다 (Candida), 토룰라 (Torula), 토룰롭시스 (Torulopsis) 및 아스페르길루스 (Aspergillus)로 구성된 군에서 선택된 세포.
- 제24항에 있어서, 비피도박테륨 브레브 (Bifidobacterium breve), 비피도박테륨 롱검 (Bifidobacterium longum), 비피도박테륨 인판티스 (Bifidobacterium infantis), 비피도박테륨 비피둠 (Bifidobacterium bifidum) 및 락토콕쿠스 락티스 (Lactococcus lactis)로 구성된 군에서 선택된 세포.
- 요구르트, 치즈, 발효시킨 우유 제품, 식이보조제 및 건강보조 (probiotic) 식품으로 구성된 군에서 선택된 제품의 생산을 위한, 제21항 내지 제25항 중 어느 한 항의 세포의 용도.
- 제21항 내지 제25항 중 어느 한 항의 세포를 포함하는 유제품.
- 갈락토-올리고당류 생산을 위한, 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소 또는 제21항 내지 제25항 중 어느 한 항의 세포의 용도.
- 요구르트, 치즈, 발효시킨 유제품, 식이보조제 및 건강보조 식품으로 구성된 군에서 선택된 제품의 일부인 갈락토-올리고당류 생산을 위한, 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소 또는 제21항 내지 제25항 중 어느 한 항의 세포의 용도.
- 비피도박테륨의 성장을 향상시키는 갈락토-올리고당류 생산을 위한, 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소 또는 제21항 내지 제25항 중 어느 한 항의 세포의 용도.
- 혼합 배양 발효물 중 비피도박테륨의 성장을 향상시키는 갈락토-올리고당류 생산을 위한, 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소 또는 제21항 내지 제25항 중 어느 한 항의 세포의 용도.
- 제21항 내지 제25항 중 어느 한 항의 세포를 적합한 배양 배지 중에서 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소의 발현을 허용하는 조건하에 배양하는 단계 및 생성된 효소를 배양물로부터 회수하는 단계를 포함하는, 제14항 내지 제18항 중 어느 한 항의 트랜스갈락토실화 효소의 생산 방법.
- 제14항 내지 제18항 중 어느 한 항의 효소 또는 제21항 내지 제25항 중 어느 한 항의 세포를 락토스 용액과 접촉시키는 단계를 포함하는, 갈락토-올리고당류의 생산 방법.
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KR1020027015920A KR100878968B1 (ko) | 2000-05-26 | 2001-05-25 | 비피도박테륨에서 단리한 베타-갈락토시다제 |
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KR (2) | KR20080045766A (ko) |
AT (1) | ATE352611T1 (ko) |
AU (2) | AU6206801A (ko) |
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ATE360682T1 (de) * | 2003-06-30 | 2007-05-15 | Clasado Inc | Neue galactooligosaccharidzusammensetzung und herstellung davon |
GB0525857D0 (en) | 2005-12-20 | 2006-02-01 | Product and process | |
GB0601901D0 (en) | 2006-01-31 | 2006-03-08 | Product and Process | |
GB0606112D0 (en) | 2006-03-28 | 2006-05-03 | Product and process | |
US8071353B2 (en) | 2006-08-18 | 2011-12-06 | Nestec S.A. | Genetic remodeling in Bifidobacterium |
US8361756B2 (en) * | 2006-09-15 | 2013-01-29 | The Regents Of The University Of California | Bifidobacterial gene sequences and their use |
WO2009071539A1 (en) | 2007-12-03 | 2009-06-11 | Novozymes A/S | Method for producing a dairy product |
GB0809921D0 (en) * | 2008-05-30 | 2008-07-09 | Clasado Inc | Product and process therefor |
US20090297660A1 (en) * | 2008-06-02 | 2009-12-03 | Kraft Food Holdings, Inc. | Cheese Products Containing Galacto-Oligosaccharides And Having Reduced Lactose Levels |
US11065268B2 (en) | 2009-05-27 | 2021-07-20 | Clasado Research Services Limited | Method of preventing diarrhoea |
US20110189342A1 (en) * | 2010-02-01 | 2011-08-04 | Jeong Hea-Seok | High-purity galactooligosaccharides and uses thereof |
US9107440B2 (en) | 2010-03-29 | 2015-08-18 | Dupont Nutrition Biosciences Aps | Polypeptides having transgalactosylating activity |
JP2017018135A (ja) * | 2010-07-19 | 2017-01-26 | アルラ フーズ アムバArla Foods Amba | ガラクトオリゴ糖含有組成物およびそれを生産する方法 |
MX348325B (es) * | 2010-07-19 | 2017-06-06 | Arla Foods Amba | Composicion que contiene galacto-oligosacarido y un metodo para producirla. |
EP2620506A1 (en) * | 2012-01-25 | 2013-07-31 | Arla Foods Amba | Method of producing a galacto-oligosaccharide-containing composition |
JP6641179B2 (ja) * | 2012-06-08 | 2020-02-05 | デュポン ニュートリション バイオサイエンシス エーピーエス | トランスガラクトシル化活性を有するポリペプチド |
KR20150093775A (ko) | 2012-12-07 | 2015-08-18 | 노쓰 캐롤라이나 스테이트 유니버시티 | 베타 헥소실-전이효소 및 이의 이용 |
CA2913393A1 (en) | 2013-05-24 | 2014-11-27 | General Mills, Inc. | Food products with yogurt whey |
AU2014363517B2 (en) * | 2013-12-11 | 2018-06-28 | International N&H Denmark Aps | A method for preparing a dairy product having a stable content of galacto-oligosaccharide(s) |
WO2015107050A1 (en) * | 2014-01-14 | 2015-07-23 | Dsm Ip Assets B.V. | Improved enzyme variants of lactase from kluyveromyces lactis |
WO2015132402A1 (en) * | 2014-03-06 | 2015-09-11 | Arla Foods Amba | Lactose-reduced milk products containing galacto-oligosaccharides and monosaccharides and a method of production |
EP3214942B1 (en) | 2014-11-07 | 2021-02-24 | DuPont Nutrition Biosciences ApS | Recombinant host cell expressing beta-galactosidase and/or transgalactosylating activity deficient in mannanase, cellulase and pectinase. |
EP3215628A1 (en) * | 2014-11-07 | 2017-09-13 | DuPont Nutrition Biosciences ApS | Method of generating a saccharide containing a galactose and a fructose moiety employing enzyme with transgalactosylating activity |
WO2017167847A1 (en) | 2016-03-31 | 2017-10-05 | Dsm Ip Assets B.V. | Production of milk with low lactose content |
EP3435771A1 (en) | 2016-03-31 | 2019-02-06 | DSM IP Assets B.V. | Enzyme composition and preparation of a dairy product with improved properties |
WO2017167849A1 (en) | 2016-03-31 | 2017-10-05 | Dsm Ip Assets B.V. | Enzyme composition and preparation of a dairy product with improved properties |
PT3228194T (pt) | 2016-04-07 | 2021-04-05 | Dsm Ip Assets Bv | Produto lácteo fermentado com diacetilo produzido com auxílio de lactase |
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EP3624593A1 (en) | 2017-05-15 | 2020-03-25 | Novozymes A/S | Glycosylated beta-galactosidase compositions having improved transgalactosylating activity |
RU2698460C1 (ru) * | 2019-03-28 | 2019-08-27 | Общество с ограниченной ответственностью "Международный Биотехнологический Центр "Генериум" | Способ получения рекомбинантной в-1,4-галактозидазы bgaa из streptococcus pneumoniae |
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US5153128A (en) * | 1990-03-16 | 1992-10-06 | Suntory Limited | Heat-resistant β-galactosyltransferase, its production process and its use |
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GB9221163D0 (en) * | 1992-10-08 | 1992-11-25 | Nobipol And Protan Biopolymer | Dna compounds |
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AU2001262068B2 (en) | 2006-07-06 |
AU6206801A (en) | 2001-12-03 |
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JP2003534007A (ja) | 2003-11-18 |
US7081355B2 (en) | 2006-07-25 |
KR100878968B1 (ko) | 2009-01-19 |
ATE352611T1 (de) | 2007-02-15 |
US6555348B2 (en) | 2003-04-29 |
WO2001090317A3 (en) | 2002-04-25 |
US20020086358A1 (en) | 2002-07-04 |
DK1283876T3 (da) | 2007-02-19 |
JP5042431B2 (ja) | 2012-10-03 |
DE60126269T2 (de) | 2007-06-21 |
DE60126269D1 (de) | 2007-03-15 |
WO2001090317A2 (en) | 2001-11-29 |
US20030162280A1 (en) | 2003-08-28 |
NZ522623A (en) | 2003-10-31 |
KR20030016275A (ko) | 2003-02-26 |
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