Trichoderma harzianum 유래 ${\beta}$-mannanase에 의한 Locust Bean Gum 가수분해 올리고당의 동정 및 Bifidobacterium spp.에 대한 생육활성

Identification and Growth Activity to Bifidobacterium spp. of Locust Bean Gum Hydrolysates by Trichoderma harzianum ${\beta}$-mannanase

  • 김유진 (경원대학교 공과대학 생명공학부 분자.식품생명공학) ;
  • 박귀근 (경원대학교 공과대학 생명공학부 분자.식품생명공학)
  • Kim, Yu-Jin (Department of Food and Bioengineering, Kyungwon University) ;
  • Park, Gwi-Gun (Department of Food and Bioengineering, Kyungwon University)
  • 발행 : 2005.12.31

초록

Trichoderma harzianum이 생산하는 ${\beta}-mannanase$의 최적 사면배지조성은 2.0% malt extract, 2.0% glucose, 0.1% peptone, 2.0% agar로, 효소생산 배지조성은 3.0% cellulose, 3.0% C.S.L, 1% $KH_2PO_4$, 0.2% $(NH_4){_2}SO_4$로 결정되어 $28^{\circ}C$, 130 rpm, 진탕배양법으로 조효소액을 생산하였다. ${\beta}-mannanase$의 최적 pH와 최적온도는 pH 4.5, $60^{\circ}C$에서 최대 상대활성을 나타내었다. Locust bean gum에 대한 효소적 가수분해 pattern을 TLC에 의해 검토한 결과 반응초기부터 반응말기까지 주요 생성물은 단당류, 중합도 4와 7의 올리고당으로 확인되었다. 중합도별 올리고당을 조제하기 위하여 activated carbon column chromatography에 250 ml/hr유속으로 tube당 20 ml씩 ethanol $0{\sim}50%$의 gradient법으로 분리 회수하였고, 분리된 각각의 올리고당 중 중합도4는 TLC $R_f$ value상으로 ${\beta}-1,4-mannotetraose$로, 중합도 7은 Methylation method에 의해 M-M-M-M-M 구조로 //G-G 동정되었다. (G-와 M-은 각각 ${\alpha}-1,6-D-galactosidic,\;{\beta}-1,4-mannosidic$ 결합을 나타냄). Bifidobacterium longum에 대한 생육활성을 기존의 MRS media와 탄소원으로 중합도 4와 7의 올리고당으로 대체하였을때 3.4배, 4.3배의 생육활성이 증가하였고, Bifidobacterium bifidum에 대해서는 1.2배, 1.1배의 낮은 활성을 나타내었다.

This study was performed to elucidate substrate specificity to the locust bean gum galactomannan by Trichoderma harzianum ${\beta}-mannanase$. The medium composition for enzyme production were determined 3% cellulose, 3% corn steep liquor, 1% $KH_2PO_4$, 0.2% $(NH_4){_2}SO_4$, and incubated for 115 hr at $28^{\circ}C$. The ${\beta}-mannanase$ exhibited maximum activity at pH 4.5 and $60^{\circ}C$. Locust bean gum galactomannan was hydrolyzed by the ${\beta}-mannanase$, and then hydrolysates separated by activated carbon column chromatography. The main hydrolysates were composed of D.P 4 and 7 galactosyl mannooligosaccharides by TLC. For the elucidate the structure of D.P 4 and 7 oligosaccharides, methylation analysis was performed. D.P 4 and 7 were identified as M-M-M-M and M-M-M-M-M (G- and M-represent ${\alpha-1,6-D-galactosidic\;and\;{\beta}-1,4-mannosidic$ linkages, respectively). //G-G To investigate the effects of locust bean gum galactosyl mannooligosaccharides on the in vitro growth of B. longum, B. bifidum, B. infantis, and B. breve, Bifidobacterium spp. were cultivated individually on the modified-MRS medium containing carbon source such as D.P 4 and 7 galactosyl mannooligosaccharides, respectively. B. longum grew up 3.4-fold and 4.3-fold more effectively by the replacement of D.P 4 and 7 galactosyl mannooligosaccharides as the carbon source in a comparasion of standard MRS.

키워드

참고문헌

  1. Dekker, R. F. H. (1983) Bioconversion of hemicellulose; aspect of hemicellulase production by Trichoderma reesei QM 9414 and enzymatic saccharification of hemicellulose. Biotechnol. Bioeng. 25, 1127-1146 https://doi.org/10.1002/bit.260250419
  2. Tipson, R. S., Horton, D. In Advances in Carbohydrate chemistry and Biochemistry. Academic Press, New York, 299
  3. Tsujisaka, Y., Hiyama, K. and Fukumoto, A. (1972) Nippon Nogeikagaku Kaishi, 43, 155
  4. Park, G. G. (1995). A new method for preparation of mannotriose from white copra meal using the enzyme system and yeast fermentation. J. Korean Soc. Food Nutr. 24, 1020-1025
  5. Gyrgy, P., Norris, R. F. and Rose, C. S. (1954) Bifidus factor. I. A variant of Lactobacillus bifidus requiring a special growth factor. Arch. Biochem. Biophys. 4, 193-198
  6. Hoffomann, K. and Bircher, J. (1969) Ver nderungen der bakteriellen Darmbesidelung nach Lactulose-gaben. Schweiz. Med. Wschr. 99, 608-613
  7. Kobayashi, Y., Echizen, R. and Mutai, M. (1984) Intestinal flora and dietary factors. Processings of the 4th RlKEN symposium on intestinal flora. Japan Scientific Societies press, Tokyo. p. 69
  8. Choi, J. Y. and Park, G. G. (2004) The growth activity of Bifidobacterium spp. by the gum hydrolysates. Kor. J. Microbiol. Biotechnol. 32, 117-122
  9. Kim, S. S. and Park, G. G. (2004) Preparation of $Gal^3Man_4$ by Bacillus sp. $\beta$-mannanase and Growth Activity to Intestinal Bacteria. J. Korean Soc. Appl. Biol. Chem. 47, 379-383
  10. Mitsuoka, T. (1982) Recent trends in research on intestinal flora. Bifidobacteria. Microflora. 1, 3-11
  11. Mitsuoka, T. (1990) Bifidobacteria and their role in human health. J. Ind. Microbiol. 6, 263-269 https://doi.org/10.1007/BF01575871
  12. Choi, J. Y. and Park, G. G. (2004) Purification of Bacillus sp. $\beta$-mannanase and the Growth Activity of Bifidobacterium spp. by Guar Gum Hydrolysates. Kor. J. Microbiol. Biotechnol. 32, 117-122
  13. Torrie, J. P., Senior, D. J. and Saddler, J. N., (1990) Production of $\beta$-mannanase by Trichoderma harzianum E58. Appl. Microbiol. Biotechnol. 34, 303-307
  14. Miller, G. L. (1959) Use of dinitrosalicylic acid regent for determination of reducing sugar. Anal. Chem. 31, 426-428 https://doi.org/10.1021/ac60147a030
  15. McCleary, B. V. (1982) Purification and properties of a mannoside mannohydrolase from guar. Carbohydrate. Res. 101, 74-92
  16. Ciucanu, I. and Kerk, F. (1984) Methylation Analysis of Oligosaccharides. Carbohydrate. Res. 131, 209-217 https://doi.org/10.1016/0008-6215(84)85242-8
  17. Shin, S. H. and Park, G. G. (In Press) Separation, Preparation and Indentification of Copra Cake Hydrolysates by Xylogone sphaerospora $\beta$-mannanase. J. Korean. Soc. Food. Sci. Nutr
  18. Paula, F. F., Humberto M. F. and Edivaldo, K. F. F.(2004) Production and characterization of hemicellulase activities from Trichoderma harzianum Strain T4. Biotechnol. Appl. Biochem. 40, 255-259 https://doi.org/10.1042/BA20030161
  19. Park, G. G. and Jang, H. K. (1992) Separation and preparation of galactosyl-mannooligosaccharides from copra galactomannan by mannanase from Penicillium purpurogenum. J. Microbiol. Biotechnol. 3, 204-208
  20. Choi, J. Y. and Park, G. G. (2004) Metabolism Activitiy of Bifidobacterium spp. by D.Ps of Konjac Glucomannan Hydrolysates. J. Korean. Soc. Food. Sci. Nutr. 33, 1186-1191