한국식품과학회지 (Korean Journal of Food Science and Technology)

한국식품과학회 (Korean Society of Food Science and Technology)
권호 표지

호알카리성 Bacillus sp. 4830이 생산하는 Streptococcus mutans 세포벽 분해효소의 분리와 특성

Production and Characteristics of Lytic Enzyme against Streptococcus mutans Cell Wall from Alkalophilic Bacillus sp. 4830

  • 김윤근 (단국대학교 식품공학과) ;
  • 배동훈 (단국대학교 식품공학과)
  • Kim, Yun-Keun (Department of Food Engineering, Dankook University) ;
  • Bai, Dong-Hoon (Department of Food Engineering, Dankook University)
  • 발행 : 2003.12.01
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초록

본 연구는 Streptococcus mutans를 특이적으로 용균하는 효소를 탐색하고 효소생산 균주의 동정을 수행하였다. 토양으로부터 5,000여주의 알칼리 내성 판주를 선별하였으며 그중 용균활성이 있는 22주를 선별하였으며 가장 용균활성이 높은 균주 1주를 선별하여 Strain No. 4830이라 명명한 후 효소의 생산 조건 및 효소의 특성을 확인한 결과 pH 5에서부터 pH 11에서 안정하였으며 pH 7.0 이상에서 Streptococcus mutans에 대하여 용균활성이 나타났다. 또한 $30^{\circ}C$ 이상에서 효소의 활성을 확인할 수 있었으며 $50^{\circ}C$에서 최대효소활성을 확인하였다. 효소의 열안정성은 $40^{\circ}C$ 이하에서는 안정한 것으로 확인되었다. 균주의 특성을 검토한 결과 균체의 지방산 조성은 중성 조건에서 생장하는 Bacillus계열과는 달리 16-C계열의 지방산함량이 상당히 높은 것을 확인하였으며 Obligate alkalophilic의 특성으로 알려진 branched 15-C 지방산인 $iso-C_{15:0}$와 $anteiso-C_{15:0}$의 함량이 매우 높은 것을 확인할 수 있었다. 또한 생화학적 특성으로 Bacillus 계열로 추정이 가능하였으며 다양한 종류의 당류에 대하여 acid를 형성하지 않은 것으로 확인되어 Strain No. 4830은 Obligate alkalophilic의 특성을 나타내었다. 또한 15% NaCl의 농도에서도 생장하는 것으로 보여 염에 대하여 내성을 갖는 것으로 나타났다. 16S rRNA 분석결과 Bacillus alcalophilus 균주와 94%의 유사성을 나타내었으나 생화학적 및 이화학적인 특성 등에서 Bacillus alcalophilus와 다른 균주로 동정되었다.

To elucidate a method of preventing dental caries, strains producing lytic enzymes were isolated and their characteristics were investigated. Among 5,00 alkalophilic strains isolated from soil, 22 strains showed lytic activity against Streptococcus mutans. Strain No. 4830, with the highest lytic activity, was selected for further study. Strain 4830 showed 94% sequence homology with the 16S rDNA sequence of Bacillus alcalophilus, but it was concluded to be different from Bacillus alcalophilus because of its biochemical characteristics. The strain was named Bacillus sp. 4830. The lytic enzyme from Bacillus sp. 4830 was purified by ethanol precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined to be 28 kDa by SDS-PAGE. The lytic enzyme was stable between pH 5.0 and pH 11 and up to $40^{\circ}C$. The optimal pH and temperature for the lytic activity was 9.0 and $50^{\circ}C$, respectively.

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참고문헌

  1. Koga, T, Sato, S., Inoue, M., Takeuchi, K, Furuta, T and Hamada, S. Role of primers in glucan synthesis by glucosyltransferases from Streptococcus mutans strain OMZI76. J. Gen. Microbiol. 129: 751-754 (1983)
  2. Ernest, N.B. Cariology, 3rd ed.. Chi Won Publishing Co., Seoul, Korea (1989)
  3. Gibbons, R.J., Berman, K.S., Berman, P., Knoettner, H. and Kapsemalis, B. Dental caries and alveola bone loss in gnotobiotic rat infected with capsule forming streptococci of human origin. Arch. Oral. BioI. 11: 549-560 (1966) https://doi.org/10.1016/0003-9969(66)90220-2
  4. Krasse, B. Human streptococci and experimental caries in hamsters. Arch. Oral. BioI. 11: 429-436 (1966) https://doi.org/10.1016/0003-9969(66)90107-5
  5. Slots, J and Taubman, M. Contemporary Oral Microbiology and Immunology. Mosby Yearbook, St. Louis, USA (1992)
  6. Stevens, J.E. and Desrocher, J. Oral ecology. Technol. Rev. 100: 48-54 (1997)
  7. Willett. Essential Dental Microbilogy. Appleton & Lamge, New York,USA (1991)
  8. Hamada, S., Gill, K and Slade, H.D. Chemical and immunological properties of the type f polysaccharide antigen of Streptococcus mutans. Infect. Immun. 14: 203-211 (1976)
  9. Baum, L., Phillips, RW and Land, M.R. Textbook of Operative Dentistry. WB. Saunders Co., PA, USA (1985)
  10. Otake, S., Makimura, M. and Kuroki, T. Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Res. 25: 438-443 (1991) https://doi.org/10.1159/000261407
  11. Murray, J.J. Fluorides and Oral Health; WHO Expert Committee on Oral Health Status and Fluoride Use. WHO Technical Report Series 846 (1994)
  12. Slade, H. D. and Slamp, W.C. Peptidoglycan composition and taxonomy of group D, E, and H streptococci and Streptococcus mutans. J. Bacteriol. 109: 691-695 (1972)
  13. Gibbons, R.J., De Stoppelaar, J.D and Harden, L. Lysozyme insensitivity of bacteria indigenous to the oral cavity of man. J. Dent. Res. 45: 877-881 (1966) https://doi.org/10.1177/00220345660450036201
  14. Yogokawa, K., Kawata, S., Nishimura, S., Ikeda, Y. and Yoshimura, Y. Mutanolysin, bacteriolytic agent for cariogenic streptococci: partial puripication and properties. Antimicrobial Agent Chemotheraphy 6: 156-165 (1974) https://doi.org/10.1128/AAC.6.2.156
  15. Hamada, S., Torii, M., Kotani, S., Masuda, N., Ooshima, T, Yokogawa, K. and Kawata, S. Lysis of Streptococcus mutans cells with mutanolysin, a lytic enzyme prepared from a culture liquor of Streptomyces globisporus 1829. Arch. Oral BioI. 23: 543-549 (1978) https://doi.org/10.1016/0003-9969(78)90268-6
  16. Horikoshi, K. and Akiba, T. Alkalophilic Microorganisms-A New Microbial world. Springer-Verlag,Tokyo, Japan (1982)
  17. Hayashi, K., Kasumi, T., Kubo, N. and Tsumura, N. Purification and characterization of the lytic enzyme produced by Streptomyces rutgersensis H-46. Agric. BioI. Chem. 45: 2289-2300 (1981) https://doi.org/10.1271/bbb1961.45.2289
  18. Miller, G.L. Use of dinitrosalicylic acid regent of determination of reducing sugar. Anal. Chem. 31: 426-428 (1959) https://doi.org/10.1021/ac60147a030
  19. Daniel, M.B. and Stuart, J.E. Protein Methods, pp. 50-55. Wiley-Liss, New York, USA (1991)
  20. Kreig, N.R. and Halt, J.G. Bergey's Manual of Systematic Bacteriology. Williams and Wilkins Co., Baltimore, MD, USA (1984)
  21. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria. 2nd ed. Williams & Wilkins, Baltimore, MD, USA (1990)
  22. Yeo. I.H.. Han. S.K. Yu. J.M. and Bai. D.H. Isolation of novel alkalophilic Bacillus alcalophilus subsp. YB380 and the caracteristics if its yeast cell wall hydrolase. J. Microbiol. Biotechnol. 8: 501-508 (1998)
  23. Tcherpakov, E. and Gutnick, D.L. Paenibacillus dendritiformis sp. nov. Marianna, Proposal for a new pattern-forming species and its localization within a phylogenetic cluster. Int. J. Syst. Bacteriol. 49: 239-246 (1999) https://doi.org/10.1099/00207713-49-1-239
  24. Yumoto, I., Yamazaki, K, Sawabe, T, Nakano, K., Kawasaki, K., Ezura, Y. and Shinano, H. Bacillus horti sp. nov., a new gramnegative alkaliphilic bacillus. Int. J. Syst. Bacteriol. 48: 565-571 (1998) https://doi.org/10.1099/00207713-48-2-565