Bacillus sp. 유래 b-Mannanase정제 및 Guar Gum 가수분해 올리고당의 Bifidobacterium spp.에 대한 증식활성
Purification of Bacillus sp. b-Mannanase and the Growth Activity of Bifidobacterium spp. by Guar Gum Hydrolysates.
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DEAE-sephadex ion exchange column chromatography에 의해 Bacillus sp. 유래 b-mannanase의 정제를 수행하여 비활성 21.57 units/ml, 정제배율 95.33을 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 38.9 kDa으로 결정되었다. 정제효소에 의해 guar gaum galactomannan을 가수분해하여 1차 activated carbon column chromatography와 2차 Sephadex G-25 gel filtration에 의해 당가수분해물을 분리 회수하여 TLC 및 FACE에 의해 주요 당가수분해물은 중합도 5와 7로 확인되었다. B. longum, B. bifidum, B. infantis, B. adolescentis, B. animalis, and B. breve의 생육활성에 대한 중합도 5와 7의 영향을 검토하기 위하여 modified-MRS 배지 상에 탄소원으로 중합도 5와 7을 대체하여 생육활성을 비교한 결과 B. longum에서는 중합도 5 galactomannooligosaccharide를 탄소원으로 대체한 경우 Standard MRS와 비교하여 10배의, 중합도 7을 처리한 경우에도 7.5배의 상대 활성을 나타내어 가장 우수한 생육활성을 나타냈었다. B. bifidum의 경우에서도 중합도 5에서 9.8배, 중합도 7에서 7.7배의 우수한 생육활성을 나타내었으며 이외에도 B. breve, B. animalis, B. infantis에 있어서도 중합도 5의 경우 2.9~5.7배의 상대 활성을 나타내었으나, B. infantis에 대한 중합도 7의 경우에서는 Standard MRS와 비교하여 0.62배로 감소하였다. 또한 중합도 5의 올리고당이 중합도 7의 올리고당보다 생육활성에 크게 기여하는 것으로 나타났다.
Abstract
Bacillus sp. b-mannanase was purified by DEAE-sephadex ion exchange column chromatography. The specific activity of the purified enzyme was 21.57 units/ml protein, representing an 95.33-folds purification of the original crude extract. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 38.9 kDa. Guar gum galactomannan was hydrolyzed by the purified b-mannanase, and then the hydrolysates was separated by activated carbon column chromatography and Sephadex G-25 gel filtration. The main hydrolysates were composed of D.P. (Degree of Polymerization) 5 and 7 galactomannooligosaccharides. To investigate the effects of guar gum galactomannooligosaccharides on in vitro growth of Bifidobacterium longum, B. bifidum, B. infantis, B. adolescentis, B. animalis, and B. breve, Bifidobacterium spp. were cultivated individually on the modified-MRS medium containing carbon source such as D.P. 5 and D.P. 7 galactomannooligosaccharides, respectively. B. longum and B. bifidum grew up 10-fold and 9.8-fold more effectively by the treatment of D.P. 5 galactomannooligosaccharides, compared to those of standard MRS medium. Especially, D.P. 5 was more effective than D.P. 7 galactomannooligosaccharide on the growth of Bifidobacterium spp.
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