Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Purification of Bacillus sp. β-Mannanase and Separation of Xanthan Gum Hydrolysate by Chromatography Methods

Bacillus sp. 유래 β-Mannanase의 정제 및 Chromatography에 의한 Xanthan Gum 가수분해물의 분리

  • 박귀근 (경원대학교 생명공학부 분자ㆍ식품생명공학)
  • Published : 2003.06.01
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Abstract

A $\beta$-mannanase of Bacillus sp. was purified by DEAE Sephacel ion exchange column chromatography. The specific activity of the purified enzyme was 17.41 units/mg protein, representing an 84.74-folds purification of the original crude extract. For the separation of two types of hydrolysates by the action of purified $\beta$-mannanase, carbon column chromatography, sephadex G-25 column chromatography and thin layer chromatography were accomplished. Main hydrolysates were D.P value 5 and 7 containing of low D.P values. By the method of FACE (Fluorophore Assisted Carbohydrate Electrophoresis), two types of hydrolysates were identified to homo type.

DEAE Sepahcel ion exchange chromatography(2.5$\times$42cm)에 의해 Bacillus sp. 유래 $\beta$-Mannanase정제를 수행하였다. 정제효소의 비활성은 17.41 units/mg로서 정제배율은 84.74배를 나타내었다. Carbon column chromatography를 이용하여 0~50%의 ethanol gradient법으로 xanthan gum의 가수분해물을 분리한 결과 fraction number 40~45 및 50~60사이에서 broad한 2개 peak의 가수분해물 pattern을 나타내었다. 가수분해물의 분리도를 확인하기 위하여 TLC를 수행한 결과 fraction No. 40~44에서는 Rf value상 중합도 5에 해당하는 가수분해물이 주축을 이루고 있는 반면 fraction No.50~55에서는 중합도 7의 가수분해물이 주축을 이루고 있음을 확인할 수 있었다. 중합도별 가수분해물의 분리도를 높이기 위해 2차 Sephadex G-25 column chromatography를 수행한 결과 fraction No. 12~15에서 중합도 7의 올리고당과 fraction No. 77~80에서 중합도 5의 가수분해물을 분리 할 수 있었고, 가수분해물의 분리도를 확인하기 위해 2차 TLC를 수행 한 결과 fraction No. 12~15에서는 중합도 7이 주축을 이루고 있으나 일부 소량의 고중합도 가수분해물이 공존하고 있는 것으로 사료되며, fraction No. 77~80에서는 분리능이 높게 중합도 5의 가수분해물이 분리되었다. 이와 같이 분리된 2개의 fractions은 FACE법에 의해 Homo type가수분해물로 동정되었다.

Keywords

References

  1. Park GG, Chang HG. 1992. Sepration and preparation of galactosylmanno-oligosaccharides from copra galactomannan by mannanase from Penicillium purpurogenum. J Microbiol Biotechnol 2: 204-208.
  2. Kim JH, Lee TK, Yang HC, Oh DK. 1997. Optimization of medium for $\beta$-mannanaseproduction by Bacillus sp. WS- 42. Kor J Appl Microbiol Biotechnol 25: 212-217.
  3. Park GG.1994. Production of mannooligosaccharides by the Penicillium purpurogenum mannanase. J Korean Soc Food Nutr 23: 509-514.
  4. Tipson RS, Horton D. 1976. ${\beta}$-1,4-Mannosidic linkage of mannan. In Advances in carbohydrate chemistry and biochemistry. Academic Press, New York. Vol 32, p 299-301.
  5. Tsujisaka Y, Hiyama K, Fukumoto. 1972. Guar gum hydrolyzing enzyme in plant. Nippon Nogeikagaku Kaishi 43: 155-160.
  6. Hishimoto Y, Fukumoto J. 1969. $\beta$-1,4-Mannosidic linkage of konjak. Nippon Nogeikagaku Kaishi 43: 317-319. https://doi.org/10.1271/nogeikagaku1924.43.317
  7. Takahashi R, Kusakabe I, Maekawa A, Suzuki T, Murakami K. 1983. Studies on mannanase of Actinomycetes. Japan J Trop Agr 27: 140-147.
  8. Dekker RFH, Richard GN. 1976. Advances in carbohydrate chemistry and biochemistry. Hemicellulases: Their occurrence, purification, properties and mode of action 32: 300-301. https://doi.org/10.1016/S0065-2318(08)60339-X
  9. Isao K, Rihei T, Satoru K, Yoshio S, Kazuo M, Akio M, Takao S. 1985. Strusture of the glucomannooligosaccharides resulting from the hydrolysis of konjac glucomannan produced by a ${\beta}$-mannanase from Streptomyces sp.. Report of Research Projecton Tropical Agricultural Resorces 4:151-161.
  10. Kobayashi Y, Echizen R, Mutai M. 1984. Intestinal flora and dietary factors. Processings of the 4th RIKEN Symposium on Intestinal flora. Japan Scientific Press, Tokyo. p 69-70.
  11. Haenel H, Bending J. 1975. Bifidobacterium role of intestinal flora. In Progresses in Food and Nutrition Science. Porgam Press p 1: 21.
  12. Shin SJ. 1988. Emerging foodborne pathogenes of public health importance. The challenge and prospects for the 21st century in verterinary science 38: 77-83.
  13. Doyle MP, Roman DJ. 1992. Recovery of Campylobacter jujuni and Campylobacter coli from inoculated foods by selective enrichment. Appl Environ Microbiol 43: 1343-1349.
  14. Tauxe RV, Hargrett-Bean N, Patton CM, Wachsmuth IK. 1988. Campylobacter isolates in the United States, 1982- 1986. Morbid Weekly Report 37: 1-13.
  15. Shin SY, Park JH. 1997. Activities of oxidative enzymes related with oxygen tolerance in Bifidobacterium sp. J Microbiol Biotechnol 7: 356-359.
  16. Lemke M, Churchill PF, Wetzel RG. 1995. Effect of substrate and cell surface hydrophobicity on phosphate utilization in bacteria. Appl Environ Mocrobiol 61: 913-919.
  17. Steeg RF, Hellemons JC, Kok AE. 1999. Synergistic actions fnisin, sublethal ultrahigh pressure, and reduced temperature on bacteria and yeast. Appl Environ Microbiol 65: 4148-4154.
  18. Park GG, Jung GH, Kobayashi H. 1999. Purification and application of earthworm $\alpha$-galactosidase by affinity chromatography. Kor J Appl Microbiol Biotechnol 27: 298-307.
  19. Park GG, Lee SY, Park BK, Ham SS, Lee JH. 1991. Characteristic features of a galactosidase from Penicillium purpurogenum. J Microbiol Biotechnol 1: 90-97.
  20. Lowry OH, Rosebrough NJ, Fan AL, Randall RJ. 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-271.
  21. Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426-428. https://doi.org/10.1021/ac60147a030
  22. McCleary BV. 1982. Purification and properties of a mannoside mannohydrolase from guar. Carbohydr Res 101: 74-92. https://doi.org/10.1016/S0008-6215(00)80796-X|