Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Purification of Bacillus sp. $\beta$-Mannanase and the Growth Activity of Bifidobacterium spp. by Guar Gum Hydrolysates.

Bacillus sp.유래 $\beta$-Mannanase 정제 및 Guar Gum가수분해 올리고당의 Bifidobacterium spp.에 대한 증식활성

  • 최준영 (경원대학교 공과대학 생명공학부 분자ㆍ식품생명공학전공) ;
  • 박귀근 (경원대학교 공과대학 생명공학부 분자ㆍ식품생명공학전공)
  • Published : 2004.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

Bacillus sp. $\beta$-mannanase was purified by DEAE-sephadex ion exchange column chromatography. The specific activity of the purified enzyme was 21.57 units/$m\ell$ 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 $\beta$-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 l0-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.

DEAE-sephadex ion exchange column chromatography에 의해 Bacillus sp. 유래 $\beta$-mannanase 의정제를 수행하여 비활성 21.57 units/$m\ell$, 정제배율 95.33을 나타내었다. SDS-PAGE에 의한 단일밴드를 확인하였고, 분자량은 38.9 kDa으로 결정되었다. 정제효소에 의해 guar gum galactomannan을 가수분해하여 1차 activated carbon column chromatography 와 2차 Sephadex G-25 gel filtration에 의해 당가수분해물을 분리 회수하여 TLC 및 FACE 에 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 배지상에 탄소원으로 중합도 5와 7을 대체하여 생육활성을 비교한 결과 B. longum 에서는 중합도 5 galactomannooligosaccharide를 탄소원으로 대체한 경우 Standard MRS와 배교하여 0.62배로 감소하였다. 또한 중합도 5의 올리고당이 중합도 7의 올리고당보다 생육활성에 크게 기여하는 것으로 나타났다.

Keywords

References

  1. Appl. Microbiol. Biotechnol. v.26 Production of β-mannosidase and β-mannanse by an alkalophilic Bacillus sp. Akino, T.;N. Nakamura;K. Horikoshi
  2. Agric. Biol. Chem. v.52 Characterization of three β-mannanse by an alkalophilic Bacillus sp. Akino, T.;W. Nakamura;K. Horikoshi
  3. Biotechnol. Bioeng. v.25 Bioconversion of hemicellulose; aspect of hemicellulase production by Trichoderma reesei QM 9414 and enzymatic saccharification of hemicellulose Dekker, R. F. H. https://doi.org/10.1002/bit.260250419
  4. Appl. Environ. Microbiol. v.63 Purification and characterization of extremely thermostablemannanase, β-mannosidase, and α-galactosidase from the hyperthermophilic eubacterium Thermotoga neaplitana 5068 Duffand, G. D.;C. M. McCutchen;P. Leduc;K. N. Parker;R. M. Kelly
  5. Mol. Biotechnol. v.5 Carbohydrate electrophoresis methods by the induced ANTs Jackson, P. https://doi.org/10.1007/BF02789060
  6. Agic. Biol .Chem. v.48 Struture of the glucomannooligosaccharides resulting from the hydrolysis of konjac glucomannan produced by a β-mannanse from Streptomyces sp. Kusakabe, I.;R. Takahashi;S. Kusama;K. Murakami;A. Maekawa;T. Suzuki
  7. Nature v.227 Cleavage of struture protein during the assembly of head Bacteriophage TA Laemmli, U. K. https://doi.org/10.1038/227680a0
  8. J. Biol. Chem. v.193 Protein measurement with the folin phenol reagent Lowry, O.;H. Rosebrough;N. J. Fan;A. L.;R. J. Randall
  9. Appl. Environ. Microbiol. v.62 Improved production of mannanase by Streptomyces lividans Marga, F.;C. Ghakis;C. Duport;R. Morosoli;D. Kluefel
  10. Carbohydr. Res. v.101 Purification and properties of a mannoside mannohydrolase from guar McCleary, B. V.
  11. Anal. Chem. v.31 Use of dinitrosalicylic acid regent for determination of reducing sugar Miller, G. L. https://doi.org/10.1021/ac60147a030
  12. Foods Biothchnol. v.4 Production of β-mannanase by an alkali-tolerant Bacillus sp. YA-14 Min, D. S.;Y. J. Chung;D. H. Bai;J. H.Yu
  13. Kor. J. Dairy Sci. v.20 Effect on the growth of Bifidobacterium spp. by transgalactooligosaccharides produced with β-galactosidase Moon, J. W.;S. H. Choi;Y. K. Shin;S. W. Lee;K. H. Kang
  14. Bifidus v.2 Effect of 4'-galactosyllactose intake on human fecal microflora Ohtsuka K.;Y. Benno;Y. K. Endo;H. Ueda;O. Ozawa;T. Mitsuoka
  15. J. Microbiol. Biotechnol. v.2 Characteristic features of an α-galactosidase from Penicillium purpurogenum Park ,G. G.;S. Y. Lee;B. K. Park;S. S. Ham;J. H. Lee
  16. Agic. Biol. Chem. v.51 Purification and some properties of β-mannanase from Penicillium purpurogenum Park, G. G.;I. Kusakabe;Y. Komatsu;H. Kobayashi;T. Tasui;K. Murakami https://doi.org/10.1271/bbb1961.51.2709
  17. J. Microbiol. Biotechnol. v.3 Separation and preparation of galactosylmannooligosaccharides from copra galactomannan by mannanase from Penicillium purpurogenum Park, G. G.;H. K. Jang
  18. Agr. Biol. Chem. v.39 Isolation and characterization of oligosaccharides from an ezymic hydrolysate of konjac glucomannan Shimahara, H.;H. Suzuki;N. Sugiyama;K. Nisizawa https://doi.org/10.1271/bbb1961.39.293
  19. Agr. Biol. Chem. v.47 Isolation and Characterization of oligosaccharides from the hydrolyzate of larch wood glucomannan with endo-β-D-mannanase Shimizu, K.;M. Ishihara https://doi.org/10.1271/bbb1961.47.949
  20. Carbohydr. Res. v.299 Isolation and quantification of alginate-derived oligouronic acids by fluorphore-assisted carbohydrate electrophoresis Shimokawa, T.;S. Toshida;I. Kusakabe;T. Takenchi;K. Murata https://doi.org/10.1016/S0008-6215(96)00333-3
  21. Agric. Biol. Chem. v.54 Purification and some properties of endo-1,4-β-mannanase from Pseudomonas sp. PT-5 Yamaura, I.;T. Matsumoto;M .Funatsu;Y. Funatsu https://doi.org/10.1271/bbb1961.54.2425
  22. Agric. Biol. Chem. v.52 Specificity of β-mannanase from Penicillium purpurogenum for konjac glucomannan Zama, M.;I. Kusakabe;G. G. Park;K. Tubaki;K. Murakami https://doi.org/10.1271/bbb1961.52.519