Purification of β-Mannanase from Xylogone sphaerospora and the Effect of Picea abies Galactosyl Glucomannan Hydrolysates on the Growth of Bifidobacterium spp.
Sephadex G-100 column chromatography에 의해 Xylogone sphaerospora 유래 β-mannanase의 정제를 수행하여 비활성 8.24 units/mL 정제배율 58.86배를 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 42 kDa으로 결정되었다. 정제효소에 의해 Picea abies Galactosyl glucomannan을 가수분해하여 activated carbon column chromatography에 의해 당가수분해물을 분리 회수하여 TLC와 FACE법 및 Timell's method에 의해 중합도 7, 8, 12 and 13으로 결정되었으며, Penicillum purpurogenum 유래 정제 β-mannanase와 α-galactosidase를 이용한 enzymatic sequential action에 의해 4가지 가수분해산물 모두 hetero type galactosyl glucomannooligosaccharides로 확인되었다. B. longum, B. bifidum, B. infantis, B. animalis, B. breve, B. adolessentis, B. auglutum의 생육활성에 대한 중합도 7, 8, 12 and 13의 영향을 검토하기 위하여 modified-MRS 배지상에 탄소원으로 중합도 7, 8, 12 and 13을 대체하여 생육활성을 비교한 결과 B. longum에서는 D.P. 7 galactosyl glucomanno-oligosaccharide를 탄소원으로 대체한 경우 표준 MRS배지와 비교하여 10.8배, D.P. 8에서 12.5배, D.P. 12에서 10.2배 D.P. 13에서 9.2배의 상대활성을 나타내어 가장 우수한 생육활성을 나타내었으며, B. bifidum의 경우에서도 D.P. 7에서 3.0배, D.P. 8에서 3.3배, D.P. 12에서 3.7배 D.P. 13에서 5.7배의 활성을 보였다.
β-Mannanase from Xylogone sphaerospora was purified by Sephadex G-100 column chromatography. The specific activity of the purified enzyme was 8.24 units/mL protein, representing an 58.86-fold 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 42 kDa. Picea abies galactosyl glucomannan was hydrolyzed by the purified β-mannanase, and then the hydrolysates were separated by activated carbon column chromatography. The main hydrolysates were composed of D.P. (degree of polymerization) 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides. To investigate the effects of Picea abies galactosyl glucomanno-oligosaccharides on the in vitro growth of Bifidobacterium longum, B. bifidum, B. animalis, B. breve, B. infantis, B. adolescentis, and B. auglutum, Bifidobacterium spp. were cultivated individually on a modified-MRS medium containing a carbon source such as D.P. 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides. B. longum propagated 10.83-fold, 12.50-fold, 10.25-fold, and 9.25-fold more effectively by the treatment of D.P. 7, 8, 12, and 13 galactosyl glucomanno-oligosaccharides, respectively, compared to those of standard MRS medium. Especially, all four sorts of galactosyl glucomannooligosaccharides were more effective in promoting the growth of B. longum than B. animalis, B. bifidum, B. breve, and B. infantis.