Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Purification and Characterization of an Antifungal Antibiotic from Bacillus subtilis LAM 97-44

Bacillus subtilis LAM 97-44가 생산하는 항진균성 항생물질의 정제 및 특성

  • Lee, No-Woon (Department of Microbiological Engineering, KonKuk University) ;
  • Kwon, Tae-Jong (Department of Microbiological Engineering, KonKuk University) ;
  • Yi, Dong-Heui (Department of Microbiological Engineering, KonKuk University)
  • 이노운 (건국대학교 공과대학 미생물공학과) ;
  • 권태종 (건국대학교 공과대학 미생물공학과) ;
  • 이동희 (건국대학교 공과대학 미생물공학과)
  • Published : 2003.05.31
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Abstract

A novel antifungal antibiotic for azole-resistant Candida albicans was purified from the culture broth of Bacillus subtilis LAM 97-44 by butanol extraction, Diaion HP-20 and Dowex-50 adsorption chromatography, silica gel flash chromatography followed by HPLC and designated LAM-44A. LAM-44A was stable for 60 min at $100^{\circ}C$, and pH range from 2 to 10. MIC values were observed at $0.5-3.5\;{\mu}g/ml$ against various Candida albicans strains. The antibiotic showed no cytotoxicity for S180, MKN-45, P388, HeLa and 373 at the concentration of 1 mg/ml. LAM-f4A was colorless powder soluble in water, methanol, ethanol, butanol and negative to ninhydrin reaction. The antibiotic had maximum absorption at 273 nm in methanol, and melting point was $202^{\circ}C$. The molecular weight and formula were determined to be 282 and $C_{14}H_{34}O_5$ by $^1H-NMR,\;^{13}C-NMR$, IR spectrum and elemental analysis.

병원에서 분리한 azole계 항진균성 항생물질에 대한 내성을 가지고 있는 Candida albicans에 대해 강한 활성을 가지는 항진균성 물질을 Bacillus subtilis LAM 97-44의 배양액으로부터 분리 정제한 후 그 특성을 조사하였다. 원심분리한 배양상등액을 butanol 추출, Diaion HP-20과 Dowex-50 adsorption chromatography, silica gel flash chromatography와 HPLC로 정제하였고 TLC와 HPLC로 확인하여 그 물질을 LAM-44A라 명명하였다. LAM-44A는 pH와 열에 매우 안정하였으며 Candida sp., Cryptococcus sp. 등에 대해 강한 활성을 나타낸 반면에 독성은 매우 적었다. 분리한 물질은 273 m에서 최대흡광도를 가진 융점 $202^{\circ}C$의 무색분말이었으며 ninhydrin 반응결과 음성이었고 $^1H-NMR$, $^{12}C-NMR$, IR spectrum, 원소분석 등의 결과로 볼 때 분자량 282의 $C_{14}H_{34}O_5$의 화학식을 가진 물질로 동정되었다.

Keywords

References

  1. Strohl, W. R. (1997) In Industrial antibiotics: Today and the future. Strohl, W. R. (ed.), Biotechnology of antibiotics (2nd ed.), Mercel Dekker, New York, pp. 3-47
  2. Lartey, P. A. and Moehle, C. M. (1997) In Recent advances in antifungal agents. Annual Reports in Medicinal Chemistry, Plattner, J. J. (ed.), Academic Press, pp. 151-160
  3. Fostel, J. M. and Lartey, P. A. (2000) Emerging novel antifungal agents, Therapeutic Focus 5, 25-32
  4. Lee, N. W., Kim, C. S., Do, J. H., Jung, I. C., Lee, H. W and Yi, D. H. (1998) Isolation and identification of Bacillus sp. LAM97-44 producing antifungal antibiotics. Agric. Chem. Biotechnol. 41, 208-212
  5. Yi, D. H. and Lee, N. W. (2000) Production conditions of Bacillus sp. LAM97-44 for a water-soluble antifungal antibiotic. J. Ind. Sci. Tech. 25, 215-229
  6. Carmichael, J., DeGraff, W G., Grzdar, A. F., Monna, J. D. and Mitchell, K. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay, assesment of chemosensitivity test. Cancer Res. 47, 936-942
  7. Kim, Y. S. and Kim, S. D. (1994) Antifungal mechanism and properties of antibiotic substances produced by Bacillus subtilis YB-70 as a biological control agent. J. Microbiol. Biotechnol. 4,296-304
  8. Lee, E. T. and Kim, S. D. (2001) Antifungal substance, 2,4diacetylphliroglucinol, produced from antagonistic bacterium Psuedomonas fluorescens 2112 against Phytophthora capsici. Kor. J. Appl. Microbiol. Biotechnol. 29, 37-42
  9. Konishi, M., Nishio, M., Saitoh, K, Miyaki, T., Oki, T. and Kawaguchi, H. (1989) Cispentacin, a new antifungal antibiotic: Production, isolation, physico-chemical properties and structure. J. Antibiotics 42, 1749-1755 https://doi.org/10.7164/antibiotics.42.1749
  10. Capobianco, J. O., Zakula, D., Coen, M. L. and Goldman, R. C. (1993) Anti-Candida activity of cispentacin: The active transport by amino acid permeases and possible mechanisms of action. Biochem. Biophys. Res. Commun. 190, 1037-1044 https://doi.org/10.1006/bbrc.1993.1153
  11. Ziegelbauer, K, Babczinski, P. and Schonfeld, W. (1998) Molecular mode of action of the antifungal beta . amino acid BAY 10-8888. Antimicrob. Agents Chemother. 42, 2197-2205
  12. Fujiu, M., Sawairi, S., Shimada, H., Takaya, H., Aoki, Y., Okuda, T. and Yokose, K. (1994) Azoxybacilin, a novel antifungal agent produced by Bacillus cereus NR2991: Production, isolation and structure elucidation. J. Antibiotics 47, 833-835 https://doi.org/10.7164/antibiotics.47.833
  13. Aoki, Y., Yamamoto, M., Hosseini-Mazinani, S. M., Koshikawa, N., Sugimoto, K and Arisawa, M. (1996) Antifungal ozocibacilin exhibits activity by inhibiting gene expression of sulfite reductase. Antimicrob. Agents Chemother. 40, 127-132
  14. Besson, F., Hourdou, M. L. and Michel, G. (1990) Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids. Biochem. Biophys. Acta. 1032, 101-106
  15. Maget-Dana, R. and Peypoux, F. (1994) Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties. Toxicology 87, 151-174 https://doi.org/10.1016/0300-483X(94)90159-7
  16. Jeong, Y. K, Shin, Y. J., Jung, M. J., Joo, W H. and Choi, J. S. (2002) Structural analysis of the antifungal antibiotic from Bacillus sp. YJ-63. Kor. J. Microbiol. Biotechnol. 30, 21-25
  17. Tenoux, I., Besson, F. and Michel, G. (1991) Studies on the antifungal antibiotics: bacillomycin D and bacillomycin D methylester. Microbios. 67, 187-193
  18. Eshita, S. M., Roberto, N. H., Beale, J. M., Mamiya, B. M. and Workman, R. F. (1995) Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the cingeners. J. Antibiotics 48, 1240-1247 https://doi.org/10.7164/antibiotics.48.1240
  19. Peypoux, F., Pommier, M. T., Marion, D., Ptak, M., Das, B. C. and Michel, G. (1986) Revised structure of mycosubtilin, a peptidolipid antibiot from Bacillus subtilis. J. Antibiotics 39, 636-641 https://doi.org/10.7164/antibiotics.39.636
  20. Besson, F. and Michel, G. (1990) Mycosubtilins B and C: minor antibiotics from mycosubtilin-producer Bacillus subtilis. Microbios. 62, 93-99