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

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

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
Download PDF
⟨ Previous Next ⟩

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 hydrolyzingenzyme 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, MurakamiK. 1983. Studies on mannanase of Actinomycetes. JapanJ 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 anddietary factors. Processings of the 4th RIKEN Symposiumon 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 publichealth importance. The challenge and prospects for the 21stcentury in verterinary science 38: 77-83.
  13. Doyle MP, Roman DJ. 1992. Recovery of Campylobacterjujuni and Campylobacter coli from inoculated foods byselective 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 substrateand cell surface hydrophobicity on phosphate utilization inbacteria. Appl Environ Mocrobiol 61: 913-919.
  17. Steeg RF, Hellemons JC, Kok AE. 1999. Synergistic actionsfnisin, sublethal ultrahigh pressure, and reduced temperatureon 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. Proteinmeasurement with the folin phenol reagent. J Biol Chem193: 265-271.
  21. Miller GL. 1959. Use of dinitrosalicylic acid reagent fordetermination 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|