Production of Extracellular Polysaccharide by Perfusion Culture of Angelica gigas Nakai Suspension Cells

배지교환식 고농도 배양에 의한 참당귀 현탁세포 유래 ECP 생산

  • Kim, Young-Hwa (Department of Biological Engineering, Inha University) ;
  • Kim, Ik-Hwan (School of Life Sciences and Biotechnology, Korea University) ;
  • Kim, Dong-Il (Department of Biological Engineering, Inha University)
  • 김영화 (인하대학교 공과대학 생물공학과) ;
  • 김익환 (고려대학교 생명공학원) ;
  • 김동일 (인하대학교 공과대학 생물공학과)
  • Published : 2006.10.30

Abstract

High-density perfusion cultivation was performed to produce extracellular polysaccharide(ECP) as immunostimulating agents in suspension cell cultures of Angelica gigas Nakai. In batch culture, the maximum cell density was 16.8 gDCW/L at day 6 and 0.9 g/L of ECP was obtained at day 8. When the medium exchange was started at the fifth day after inoculation for the perfusion culture, high concentration of the cells at 23.8 gDCW/L could be achieved with continuous production of ECP. Treatments of ultrasound and Pluronic F-68 were found to be helpful for the secretion of intracellular ECP into the culture medium.

본 연구에서는 참당귀 현탁세포배양에 의한 면역 증강성 ECP의 생산을 증진시키기 위하여 배지교환식 배양을 수행하였으며, ECP의 분비를 촉진하는 초음파 처리와 Pluronic F-68의 영향을 조사하였다. 참당귀의 현탁세포배양시, 최대 세포생장은 6일째에 16.8 g DCW/L였고, ECP의 생산은 세포생장과 함께 증가하다가 8일째에 최고 0.9 g/L가 생산되었다. 식물세포 고농도배양법인 배지교환식 배양을 적용해, 초기 당 농토를 높이고 접종 후 5일부터 연속적으로 배지교환을 해주어 23.8 gDCW/L의 고농도의 현탁세포를 얻을 수 있었다. 초음파 처리 및 Pluronic F-68의 첨가는 참당귀 세포의 세포막의 투과성을 증진시켜 ECP의 생산성을 높임과 동시에 세포 내의 다당의 배지로의 배출을 유도한 것으로 판단된다.

Keywords

References

  1. Han, S. B., Y. H. Kim, C. W. Lee, S. M. Park, H. Y. Lee, K. S. Ahn, I. -H. Kim and H. M. Kim (1998), Characteristic immunostimulation by angelan isolated from Angelica gigas Nakai, Immunopharmacology 40, 39-48 https://doi.org/10.1016/S0162-3109(98)00026-5
  2. Ahn, K. S. (1996), A study on the anticancer and immunostimulation agents from the root of Angelica gigas Nakai, Ph.D. Dissertation, Dept. of Biology, Korea University, Seoul
  3. Endress, E. (1994), Plant Cell Biotechnology, Springer-Verlag, Berlin
  4. Ghosh, A. C., S. Ghosh, and P. S. Basu (2005), Production of extracellular polysaccharide by a Rhizobium species from root nodules of the Leguminous tree Dalbergia lanceolaria. Eng. Life Sci. 5, 378-382 https://doi.org/10.1002/elsc.200500087
  5. Melchart, D., C. Clemm, B. Weber, T. Draczynski, F. Worku, K. Linde, W. Weidenhammer, H. Wagner, and R. Saller (2002), Polysaccharides isolated from Echinacea purpurea herba cell cultures to counteract undesired effects of chemotherapy - a pilot study, Phytother. Res. 16, 138-142 https://doi.org/10.1002/ptr.888
  6. Roberts, S. C. and M. L. Shuler (1997), Large scale plant cell culture, Curr. Opin. Biotechnol. 8, 154-159 https://doi.org/10.1016/S0958-1669(97)80094-8
  7. Griffiths, J. B., D. Looby, and A. J. Racher (1992), Maximisation of perfusion system and process comparison with batch-type cultures, Cytotechnol. 9, 3-9 https://doi.org/10.1007/BF02521726
  8. Velez, D., L. Miller, and J. D. Macmillan (1989), Use of tangential flow filtration in perfusion propagation of hybridoma cells for production of monoclonal antibodies, Biotechnol. Bioeng. 33, 938-940 https://doi.org/10.1002/bit.260330721
  9. Su, W. W. and A. E. Humphrey (1991), Production of rosmarinic acid from perfusion culture of Anchusa officinalis in membrane-aerated bioreactor, Biotechnol. Lett. 13, 889-892 https://doi.org/10.1007/BF01022093
  10. Su, W. W., F. Lei, and N. P. Kao (1995), High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration, Appl. Microbiol. Biotechnol. 44, 293-299 https://doi.org/10.1007/BF00169919
  11. Deng, C. X., F. Sieling, H. Pan, and J. Cui (2004), Ultrasound-induced cell membrane porosity, Ultrasound Med. Biol. 30, 519-526 https://doi.org/10.1016/j.ultrasmedbio.2004.01.005
  12. Liu, Y., A. Yoshikoshi, B. Wang, and A. Sakanishi (2003), Influence of ultrasonic stimulation on the growth and proliferation of Oryza sativa Nipponbare callus cells, Colloids Surfaces B: Biointerfaces 27, 287-293 https://doi.org/10.1016/S0927-7765(02)00052-8
  13. Mutwakil, M. H. A. Z., T. J. G. Steele, K. C. Lowe, and D. I. de Pomeral (1997), Surfactant stimulation of growth in the nematode Caenorhabditis elegans, Enzyme Microb. Technol. 20, 462-470 https://doi.org/10.1016/S0141-0229(96)01173-8
  14. Bassetti, L. and J. Tramper (1995), Increased anthraquinone production by Morinda citrifolia in a two-phase system with Pluronic F-68, Enzyme Microb. Technol. 17, 353-358 https://doi.org/10.1016/0141-0229(94)00059-X
  15. Lin, L. D., J. Y. Wu, K. P. Ho, and S. Y. Qi (2001), Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures, Ultrasound Med. Biol. 27, 1147-1152 https://doi.org/10.1016/S0301-5629(01)00412-4
  16. Lin, L. D. and J. Y. Wu (2002), Enhancement of shikonin production in single and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound, Biotechnol. Bioeng. 78, 81-88 https://doi.org/10.1002/bit.10180