생명과학회지 (Journal of Life Science)

  • 제18권1호
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  • Pages.52-57
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  • 2008
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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Bacillus subtilis 유래 재조합 endoxylanase를 이용한 xylooligosaccharide의 최적 생산

Optimal Production of Xylooligosaccharide by Using Recombinant Endoxylanase from Bacillus subtilis

  • 김연희 (동의대학교 생명공학과) ;
  • 허선연 (한국생명공학연구원 분자생물공정 연구센터) ;
  • 김미진 (동의대학교 바이오물질제어학과) ;
  • 이재헝 (동의대학교 바이오물질제어학과) ;
  • 김영만 (동의대학교 식품영양학과) ;
  • 남수완 (동의대학교 생명공학과)
  • Kim, Yeon-Hee (Dept. Biotechnology & Bioengineering, Dong-Eui University) ;
  • Heo, Sun-Yeon (Molecular Bioprocess Research Center, KRIBB) ;
  • Kim, Mi-Jin (Dept. Biomaterial Control, Dong-Eui University) ;
  • Lee, Jae-Hyung (Dept. Biomaterial Control, Dong-Eui University) ;
  • Kim, Young-Man (Dept. Food Science & Nutrition, Dong-Eui University) ;
  • Nam, Soo-Wan (Dept. Biotechnology & Bioengineering, Dong-Eui University)
  • 발행 : 2008.01.31
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초록

식물체 구성성분의 하나인 hemicellulose의 대부분을 차지하는 xylan은 농산 폐기물 또는 목재 성분의 약 30%를 차지하는 풍부한 자원물질이다. 이러한 xylan을 효과적으로 분해하기 위해 Bacillus에서 발현시킨 재조합 endoxylanase를 이용하여 기능성 식품소채인 xylooligosaccharide의 최적 생산 조건을 조사하였다. B. subtilis 재조합 균주 DB431/pJHKJ4를 이용한 발효조 회분배양 결과, endoxylanse의 총 활성은 857 unit/ml이며, 대부분이 세포밖으로 분비됨을 알 수 있었고, 분비효율은 92%로 나타났다. 재조합 endoxylanase를 이용하여 xylan으로부터 xylooligosaccharide 생성을 위한 최적 반응조건을 검토한 결과, xylooligosaccharide 생산을 위한 기질로는 birchwood xylan이 적합함을 알 수 있었고, 4%의 xylan 농도에서 가장 많은 xylooligosaccharide 을 생산하며, xylobiose와 xylotriose가 주생성물임을 알았다. 효소의 농토와 반응시간의 영향은 10 unit의 endoxylanase를 첨가하여 1시간 반응시켰을 때 가장 많은 양의 xylooligosaccharide을 생산할 수 있었고, 반응온도는 $40^{\circ}C{\sim}50^{\circ}C$가 적합함을 알았다. 결론적으로 재조합 endoxylanase을 이용한 xylooligosaccharide생성에는 10 unit endoxylanase과 4% birchwood xylan을 기질로 이용하여 $50^{\circ}C$에서 1시간 반응시키는 것이 최적반응 조건임을 알았다.

Xylan is a major hemicellulose component of the cell walls of monocots and hardwood, representing up to 30% of the dry weight of these plants. To efficiently hydrolyze xylan, the endoxylanase gene from Bacillus sp. was expressed in B. subtilis DB431 by introducing the plasmid pJHKJ4. The total activity of the recombinant endoxylanase reached about 857 unit/ml by batch fermentation of B. subtilis DB431/pJHKJ4 in LB maltose medium. The majority (>92%) of endoxylanase was efficiently secreted into the culture medium. The recombinant endoxylanase hydrolyzed more the birchwood xylan efficiently than the other xylans. When 4 % concentration of xylan was used, the highest production of xylooligosaccharide was observed, and xylobiose and xylotriose were the major products. Optimal amount of enzyme and reaction time for producing xylooligosaccharide were found to be 10 unit and 1 hr, respectively. In addition, the temperature of $40^{\circ}C{\sim}50^{\circ}C$ gave the highest production of xylooligosaccharide. Consequently, the optimized conditions for the production of xylooligosaccharide through the hydrolysis of xylan were determined as follows: 10 unit endoxylanase, $50^{\circ}C$, 4% birchwood xylan, 1 hr reaction.

키워드

참고문헌

  1. Bajpai, P. 1999. Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147-157. https://doi.org/10.1021/bp990013k
  2. Beg. Q. K., M. Kapoor, L. Mahajan and G. S. Hoondal. 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56, 326-338. https://doi.org/10.1007/s002530100704
  3. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286-290. https://doi.org/10.1016/0167-7799(85)90004-6
  4. Chun, Y. C., K. H. Jung, J. C. Lee, S. H. Park, H. K. Chung and K. H Yoon, 1998. Molecular cloning and the nucleotide sequence of a Bacillus sp. KK-1 $\beta$-xylosidase gene. J. Microbiol. Biotechnol. 8, 28-33.
  5. Choi, J. H, O. S. Lee, J. H. Shin, Y. Y. Kwak, Y. M. Kim and I. K. Rhee. 2006. Thermostable xylanase encoded by xynA of Streptomyces thermocyaneoviolaceus; cloning, purification, characterization and production of xylooligosaccharides. J. Microbiol. Biotechnol. 16, 57-63.
  6. Contente, S. and D. Dubnau. 1979. Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. Mol. Gen. Genet. 167, 251-258. https://doi.org/10.1007/BF00267416
  7. Degnan, B. A. and G. T. Macfarlane. 1991. Comparison of carbohydrate substrate preferences in eight species of Bifidobacteria. FEMS Microbiol. Lett. 68, 151-156.
  8. Gupta. N., V. S. Reddy, S. Maiti and A. Ghosh. 2000. Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. Strain NG-27. Appl. Environ. Microbiol. 66, 2631-2635. https://doi.org/10.1128/AEM.66.6.2631-2635.2000
  9. Heo S. Y., J. K. Kim, Y. M. Kim and S. W. Nam. 2004. Xylan hydrolysis by treatment with endoxylanase and $\beta$-xylosidase expressed in yeast. J. Microbiol. Biotechnol. 14, 171-177.
  10. Jeong, K. J., I. Y. Park, M. S. Kim and S. C. Kim. 1998. High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan. Appl. Microbiol. Biotechnol. 50, 113-118. https://doi.org/10.1007/s002530051264
  11. Jeong, K. J., P. C. Lee, I. Y. Park, M. S. Kim and S. C. Kim. 1998. Molecular cloning and characterization of an endoxylanase gene of Bacillus sp. in Escherichia coli. Enz. Microb. Technol. 22, 599-605. https://doi.org/10.1016/S0141-0229(97)00256-1
  12. Johnvesly, B., S. Virupakshi, G. N. Patil, Ramalingam and G. R. Naik. 2002. Cellulase -free thermostable alkaline xylanase from thermophilic and alkalophilic Bacillus sp. JB-99. J. Microbiol. Biotechnol. 12, 153-156.
  13. Kim, J. H, J. H Kim, S. C. Kim and S. W. Nam. 2000. Constitutive overexpression of endoxylanase gene in Bacillus subtilis. J. Microbiol. Biotechnol. 10, 551-553.
  14. Lee, J. J. 1995. Characterization of an endoxylanase produced by isolated Bacillus sp. M. S. Thesis, KAIST, Taejon, KOREA.
  15. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428. https://doi.org/10.1021/ac60147a030
  16. Neu, H. C. and L. A. Heppel. 1965. The release of enzymes from E. coli by osmotic shock and during the formation of spheroplast. J. Biol. Chem. 240, 3685-3692.
  17. Niehaus, F., C. Bertoldo, M. Kahler and G. Antranikian. 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51, 711-729. https://doi.org/10.1007/s002530051456
  18. Okazaki, M., S. Fujikawa and N. Matsumoto. 1990. Effect of xylooligosaccharides on growth of Bifidobacterium. J. Jpn. Soc. Nutr. Food Sci. 43, 395-401. https://doi.org/10.4327/jsnfs.43.395
  19. Petschow, B. W. and R. D. Talbott. 1990. Growth promotion of Bifidobacterium species by whey and casein fractions from human and bovine milk. J. Clin. Microbiol. 28, 287-292.
  20. Reilly, P. J. 1981. Xylanase: structure and function. Basic Life Sci. 18, 111-129.
  21. Saha. B. C. and M. A. Cotta. 2006. Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw. Biotechnol. Prog. 22, 449-453. https://doi.org/10.1021/bp050310r
  22. Srinivasan, M. C. and M. V. Rele. 1999. Microbial xylanases for paper industry. Curr. Sci. 77, 137-142.
  23. Viikari, L., A. Kantelinen, J. Sandquist and M. Linco. 1994. Xylanases in bleaching: from an idea to the industry, FEMS Microbiol. Rev. 13, 338-350.
  24. Whistler, R. L. and E. L. Richard. 1970. Hemicellulose. pp. 447-469, Pigman W, Horton D (eds), The carbohydrates. Academic Press, New York.
  25. Wong, K. K. Y., S. L. Nelson, and J. N. Saddler. 1996. Xylanase treatment for the peroxide bleaching of oxygen delignified kraft pulps derived from three softwood species. J. Biotechnol. 48, 137-145. https://doi.org/10.1016/0168-1656(96)01502-7
  26. Wong, K. K. Y., U. L. Larry and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganism: functions and applications. Microbiol. Rev. 52, 305-317.

피인용 문헌

  1. Thermostability and xylan-hydrolyzing property of endoxylanase expressed in yeast Saccharomyces cerevisiae vol.14, pp.5, 2009, https://doi.org/10.1007/s12257-009-0014-2