Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Isolation and Characterization of High Viscosity Polysaccharide Producing Endophytic Bacteria from Pueraria Root

고점도 다당류를 생산하는 갈근 내생균의 분리 및 특성

  • Whang, Kyung-Sook (Department of Biotechnology and Institute of Microbial Ecology and Resources, Mokwon University) ;
  • Choi, Seung-Hyun (Chung Mi Bio Co., Ltd.) ;
  • Han, Song-Ih (Department of Biotechnology and Institute of Microbial Ecology and Resources, Mokwon University)
  • 황경숙 (목원대학교 생명산업학부, 미생물생태자원연구소) ;
  • 최성현 (청미바이오 주식회사) ;
  • 한송이 (목원대학교 생명산업학부, 미생물생태자원연구소)
  • Published : 2007.12.30
Download PDF
⟨ Previous Next ⟩

Abstract

Fifty endophytic bacteria, which produced slime around the colonies, were isolated from Pueraria roots. In particular, HDN-14, TDG-3, and TNB-3 strains, which appeared to be high viscosity producers, were selected. These strains produced high levels of polysaccharides in Puerara root medium extract. The purified polysaccharide was digested with 1N HCI and analyzed by HPLC, with glucose ($45.6{\sim}63.1%$), maltose ($14.6{\sim}23.7%$), and fructose ($17.4{\sim}23.7%$) detected as constitutive sugars. When determined by the homology relationship of the 16S rDNA sequence with the relative taxa, the HDN-14 and TNB-3 strains were closely ($99.06{\sim}99.32%$) related to the Pseudomonas $koreensis^T$ and Pseudomonas $jessenii^T$, while TDG-3 were closely ($99.48{\sim}99.74%$) related to Pseudomonas $plecoglossicida^T$, Pseudomonas $mosselii^T$, and Pseudomonas $monteilii^T$. The major cellular Pseudomonas acids are $3OH-C_{10:0}$, $2OH-C_{12:0}$, $3OH-C_{12:0}$, and $3OH-C_{12:1}$, with these strains being further differentiated in species belonging to the genus Pseudomonas.

식용 및 약용으로 광범위하게 이용되고 있는 갈근 내부조직으로부터 강한 점액성 물질을 생산하는 50개의 내생 균주를 분리하였다. 다당류의 수율이 높은 내생균주 HDN-14, TDG-3및 TNB-3 균주를 선발하여 이들이 생산하는 다당류의 구성당 조성을 분석한 결과 glucose ($45.6{\sim}63.1%$), maltose ($14.6{\sim}23.7%$) 및 fructose ($17.4{\sim}23.7%$)가 검출되었다. 이들 분리균주의 16S rDNA 염기서열(약 1,300bp)을 결정하여 계통학적 위치를 검토한 결좌, ${\grmma}$-Proteobacteria 그룹의 Pseudomonas 속에 속하는 균주임이 확인되었으며, HDN-14과 TNB-3 균주는 Pseudomonas $koreensis^T$, Pseudomonas $jessenii^T$와 $99.06{\sim}99.32%$의 상동성을 나타내었으며 TDG-3 균주는 Pseudomonas $plecoglossicida^T$, Pseudomonas $mosselii^T$, Pseudomonas $monteilii^T$와 $99.48{\sim}99.74%$ 상동성을 나타내었다. 이들 분리균주는 주요 균체지방산으로 $3OH-C_{10:0}$, $2OH-C_{12:0}$, $3OH-C_{12:0}$, $3OH-C_{12:0}$ 및 $3OH-C_{12:1}$가 검출되었으며, TDG-3 균주의 경우, 3-OH $C_{13:0}$, 3-OH $C_{14:0}$, 3-OH $C_{15:0}$ 및 3-OH $C_{16:0}$등 다양한 종류의 3-OH 지방산이 검출되어 기존의 Pseudomonas 종과 비교하여 매우 특징적인 성질을 나타내었다.

Keywords

References

  1. 엄안흠, 어주경, 김동훈, 정현숙. 2004. 18S rDNA를 이용한 인삼(panax inseng)의 내생균근 균의 동정. 한국응용생명화학회지 47, 182-186
  2. 이재훈. 1995. 미생물 다당류의 다양성과 산업적 이용 및 전망. 생물산업 8, 4033-4043
  3. 최재을, 육진아, 김진희, 최춘환, 천종식, 김영준, 이향범. 2005. 적변삼으로부터 분리한 내생세균의 동정 및 적변 유발. 한국약용작물학회지 13, 1-5
  4. 황경숙, 최성현, 조민혜. 2003. Pseudomonas sp. nov. HDN-14 갈근내생균의 분리 및 특성. 목원대학교 자연과학연구소 제 12권, 1호
  5. Goubet, F., P. Jackson, M.J. Deery, and P. Dupree. 2002. Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal. Biochem. 300, 53-68 https://doi.org/10.1006/abio.2001.5444
  6. Hallmann, J., A. Quadt-Hallmann, W.F. Mahaffee, and J.W. Kloepper. 1997. Bacterial endophytes in agricultural crops. Can. J. Microbiol. 43, 895-914 https://doi.org/10.1139/m97-131
  7. Hayagawa, J., N. Noda, S. Yamada, and K. Uno. 1984. Studies on physical and chemical quality evaluation of crude drugs preparations. I. Analysis of pueraria radix and species puerariae. J. Pharm. Soc. Jpn. 104, 50-56 https://doi.org/10.1248/yakushi1947.104.1_50
  8. Heng, Z., Q. Feng, and H. Zhu. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Res. 21, 5279-5280 https://doi.org/10.1093/nar/21.22.5279
  9. Kirchhof, G., B. Eckert, M. Stoffels, J.I. Baldani, V.M. Reis, and A. Hartmann. 2001. Herbaspirillum frisingense sp. nov., a new nitrogen- fixing bacterial species that occurs in C4-fibre plants. Int. J. syst. Evol. Microbiol. 51, 157-168 https://doi.org/10.1099/00207713-51-1-157
  10. Leong, J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogenes. Annu. Rev. Phyotopathol. 73, 217-219
  11. Linker, A. and R.S. Jones. 1966. A new polysaccharide resembling alginic acid isolated from Pseudomonas. J. Biol. Chem. 241, 3845
  12. Margaritis, A. and N.G.W. Pace. 1985. Comprehensive biotechnology: The principles, applications, and regulations of biotechnology in industry, agriculture, and medicine. Microbial Polysaccharide 3, 1005-1043
  13. Nowak, J. 1998. Benefits of in vitro 'biotization' of plant tissue cultures with microbial inoculants. In Vitro Cell. Dev. Biol. Plant. 34, 122-130 https://doi.org/10.1007/BF02822776
  14. Ohshima, Y., T. Okuyama, K. Takahashi, T. Takakizawa, and S. Shibata. 1988. Isolation and high performance liquid chromatography (HPLC) of isoflavonoids from the Pueraria root. Planta Med. 54, 250-254 https://doi.org/10.1055/s-2006-962420
  15. Oyaiz, H. and K. Komagata 1983. Grouping of Pseudomonas species on the basis of cellular fatty acid composition and the quinone system with special reference to the existence of 3-hydroxy fatty acids. J. Gen. Appl. Microbiol. 29, 17-40 https://doi.org/10.2323/jgam.29.17
  16. Reiter, B., U. Pfeifer, H. Schwab, and A. Sessitsch. 2002. Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica. Appl. Environ. Microbiol. 68, 2261-2268 https://doi.org/10.1128/AEM.68.5.2261-2268.2002
  17. Ruijssenaars, H.J., F. Stingele, and S. Hartmans. 2000. Biodegradability of food-associated extracellular polysaccharides. Curr. Microbiol. 40, 194-199 https://doi.org/10.1007/s002849910039
  18. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  19. Schiessendoppler, E. and P. Cate. 1996. Schwarzbeinigkeit, bakterille stengelfaule und Nassfaute der knolle. In B. Zwatz and Bedlan (ed.), Wichtige Krankheiten und Schadlinge der Kartoffel. p. 48-51. Institut fur Phytomedizin im Bundesamt und Forschungszentrum fur Landwirschaft, Vienna, Austria
  20. Sharma, V.K. and J. Novak. 1998. Enhancement of verticillium wilt. resistance in tomato transplants by in vitro co-culture of seedlings. with a plant growth promoting rhizobacterium (Pseudomonas sp. strain PsJN). Can. J. Microbiol. 44, 528-536 https://doi.org/10.1139/cjm-44-6-528
  21. Shibata, S., T. Murakami, and Y. Nishikawa. 1959. Studies on the constituents of Japanese Chinese crude drugs. I. On the constituents of Puerariea root. 葯學雜志 79, 757-760
  22. Stead, D.E. 1992. Grouping of plant-pathogenic and some other Pseudomonas spp. by using cellular fatty acid profiles. Int. J. Syst. Bacteriol. 42, 281-295 https://doi.org/10.1099/00207713-42-2-281
  23. Stoltzfus, J.R., R. So, P.P. Malarvithi, J.K. Ladha, and F.J. De Bruijin. 1998. Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194, 25-36
  24. Sturz, A.V., B.R. Christie, B.G. Matheson, W.J. Arsenault, and N.A. Buchanan. 1999. Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogenes. Plant Pathol. 48, 360-369 https://doi.org/10.1046/j.1365-3059.1999.00351.x
  25. Thomson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  26. Yalpani, M. and A. Sandford. 1987. Commercial polysaccharides : Recent trends and developments.Industrial polysaccharides: genetic engineering, Structure/Property relation and applications. Edited by Yalpani, M. Amsterdam-printed in the Netherlands: Elsevier Science Publishers 311-335