초록 ▼

Ⅳ. 연구개발결과
선발균주에 대한 탄소원의 종류 등 다양한 배양조건의 최적화를 통하여 효소생산을 극대화하였으며 이들 효소의 반응조건을 탐색하여 최적의 HMO 생산 조건을 확립하였음. Intracellular로 생산되는 HMO 생산용 효소의 효과적인 회수를 위하여 세포파쇄조건을 확립하여 대량생산을 위한 기초를 확립하였음. 이때 생산된 HMO를 fraction별로 정제하여 각각의 HMO를 maldi-Tof를 통하여 동정하였으며 선발균주 유래의 α, β-galactosidase 유전자를 클로닝 및 발현하여 정제하여 효소특성을 규명하

Ⅳ. 연구개발결과
선발균주에 대한 탄소원의 종류 등 다양한 배양조건의 최적화를 통하여 효소생산을 극대화하였으며 이들 효소의 반응조건을 탐색하여 최적의 HMO 생산 조건을 확립하였음. Intracellular로 생산되는 HMO 생산용 효소의 효과적인 회수를 위하여 세포파쇄조건을 확립하여 대량생산을 위한 기초를 확립하였음. 이때 생산된 HMO를 fraction별로 정제하여 각각의 HMO를 maldi-Tof를 통하여 동정하였으며 선발균주 유래의 α, β-galactosidase 유전자를 클로닝 및 발현하여 정제하여 효소특성을 규명하였음.
선발균주의 고유성을 확보하기 위하여 전체 게놈에 대한 분석을 실시하였으며 이를 통하여 bifidogenic 활성 관련 유전자의 클로닝에 활용하였음.
본 연구를 통하여 생산된 HMO의 B. bifidum BGN4에 대한 선택적인 bifidogenic 활성을 확인하였으며 유전자 수준에서 그 메커니즘을 규명하였음.
기초 연구내용을 산업적 생산규모에서 스케일업 연구를 통하여 대량생산을 확인하였으며 이를 이용하여 효소, HMO, 생균제 등의 시제품을 완성하였음.

Abstract ▼

Human milk oligosaccharides (HMOs) are known to be beneficial to infant intestinal microorganisms and provide protection against pathogens. HMOs are selectively fermented and stimulate the growth and activity of some intestinal microbiota which benefit the health of the host. Among infant intestinal

Human milk oligosaccharides (HMOs) are known to be beneficial to infant intestinal microorganisms and provide protection against pathogens. HMOs are selectively fermented and stimulate the growth and activity of some intestinal microbiota which benefit the health of the host. Among infant intestinal bacteria such as bifidobacteria have the ability to catalyze HMOs. Up to 200 different structures of HMO have been identified in the human breast milk. All HMOs follow the same basic configuration: a lactose core at the reducing end, elongated by N-acetyl-lactosamine units with at least twelve different types of glycosidic bonds, wherein fucose and sialic acid residues are added to terminal positions. The most abundant oligosaccharides among HMOs are fucosylated and the proportion of fucosylation is approximately 77%. There have been various efforts to efficiently produce HMO without no promising results. Some tried to use recombinant E. coli system which requires expensive substrates and tedious purification steps. During our previous study, we have isolated Bifidobacterium longum RD47 which showed fucosyl and galactosyl transferase activity and produced various galactooligosaccharide including fucosylated ones. In this research, we have optimized enzyme production conditions for α- and β-galactosidases through optimization of medium and culture condition of this strain. The corresponding genes have been cloned, expressed and purified and characterized for their activity at various conditions and substrates. The full genome sequence of the RD47 was obtained through re-sequencing and applied for the cloning and characterization of this strain. The crude and purified enzyme was used for the production of HMO and the product was fractionated through prep-HPLC and identified each HMO using Maldi-TOF. For the efficient preparation of the intracellular HMO producing enzymes of RD47, cell disruption and extraction process was investigated and optimized. The selective bifidogenic activity of the produced HMO was confirmed using probiotic B. bifidum BGN4 and the mechanism was elucidated at the genetic level. All of the optimized conditions of benchtop level was scale-up to the production scale and several sample product including enzyme, HMO and probiotics were successfully produced.