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Characteristics and Partial Purification of a Bacteriocin Produced by Pediococcus damnosus JNU 534

Pediococcus damnosus JNU 534가 생산하는 박테리오신의 특성 및 정제

  • Lee, Jae-Won (Division of Animal Science, Chonnam National University) ;
  • Han, Su-Min (Division of Animal Science, Chonnam National University) ;
  • Yun, Bo-Hyun (Division of Animal Science, Chonnam National University) ;
  • Oh, Se-Jong (Division of Animal Science, Chonnam National University)
  • 이재원 (전남대학교 동물자원학부) ;
  • 한수민 (전남대학교 동물자원학부) ;
  • 윤보현 (전남대학교 동물자원학부) ;
  • 오세종 (전남대학교 동물자원학부)
  • Received : 2011.10.23
  • Accepted : 2011.12.12
  • Published : 2011.12.31

Abstract

A new bacteriocin-producing lactic acid bacteria (LAB) which has been isolated from kimchi was identified as Pediococcus damnosus by use of API kit and 16S rDNA sequencing, and designated as P. damnosus JNU 534. The bacteriocin produced by P. damnosus JNU 534 markedly inhibited the growth of some of LAB and Listeria monocytogenes, whereas other pathogens including Gram negative bacteria were not susceptible. The production of bacteriocin started at the beginning of exponential phase and reached maximum activity at the early stationary phase. The bacteriocin was stable on the wide pH range of 2-9 and heat treatment up to $100^{\circ}C$ for 15 min. The antimicrobial compound was inactivated by treatments of proteolytic enzymes indicating its proteinaceous in nature. The bacteriocin was purified by 30% ammonium sulfate precipitation followed by hydrophobic interaction column and $C_{18}$ column chromatography. The estimated molecular weight of the bacteriocin using tricine SDS-PAGE was approximately 3.4 kDa and the identified N-terminal amino acid sequence was $NH_2$-ILLEELNV.

여러 원천에서 분리한 유산균을 대상으로 신규 bacteriocin 생산 유산균을 선발하고 API 당 발효성과 16S rDNA를 분석한 결과 P. damnosus 로 동정되어 JNU 534로 명명하였다. P. damnosus JNU 534에 의해 생산된 bacteriocin은 다수의 유산균과 L. monocytogenes의 생육을 억제하였지만, Gram음성균을 포함한 다른 병원성균들은 저항성을 나타냈다. Bacteriocin의 생산은 대수기 초반에 시작되어 정지기 초기에 최대 활성에 이르렀다. Bacteriocin은 pH 2-9의 넓은 범위와 $100^{\circ}C$에서 15분간의 열처리에 대해서도 안정성을 유지하였다. 또한 각종 단백질 분해효소에 의해 활성을 상실함으로써 본 연구에서 정제한 항균 물질이 단백질성 물질임을 확인할 수 있었으며 30% ammonium sulfate 침전과 $C_{18}$ chromatography를 통하여 bacteriocin을 정제 할 수 있었다. Tricine-SDS-PAGE에 의해 bacteriocin의 분자량을 약 3.4 kDa으로 추정하였으며, 지시균의 생육 저지환의 위치가 bacteriocin band와 일치하였다. 정제된 bacteriocin으로부터 분석한 N-terminal 아미노산 부분 서열은 $NH_2$-ILLEELNV로 나타났다.

Keywords

References

  1. Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Boyaval, P., and Marion, D. (1996) Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity. Appl. Environ. Microbiol. 62, 4410-4416.
  2. Bhunia, A. K., Johnson, M. C., and Ray, B. (1987) Direct detection and antimicrobial of Pediococcus acidilatici in sodium dodecyl sulfate-polyacryamide gel electrophoresis. J. Ind. Microbiol. Biotchnol. 2, 319-322. https://doi.org/10.1007/BF01569434
  3. Bhunia, A. K., Johnson, M. C., Ray, B., and Kalchayanand, N. (1991) Mode of action of pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains. J. Appl. Bacteriol. 70, 25-33. https://doi.org/10.1111/j.1365-2672.1991.tb03782.x
  4. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, B., Orecchia, P., Zardi, L., and Righetti, P. G. (2004) Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25, 1327-1333. https://doi.org/10.1002/elps.200305844
  5. Elegado, F. B. and Kwon, D. Y. (1998) Primary structure and conformational studies of pediocin AcM, a bacteriocin from Pediococcus acidilactici M. Phil. J. Biotechnol. 9, 19-26.
  6. Elegado, F. B., Kim, J. W., and Kwon, D. Y. (1997) Rapid purification, partial characterization and antimicrobial spectrum of the bacteriocin, pediocin AcM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 37, 1-11. https://doi.org/10.1016/S0168-1605(97)00037-8
  7. Ennahar, S., Sashihara, T., Sonomoto, K., and Ayaaki, I. (2000) Class II a bacteriocins: biosynthesis, structure and activity. FEMS Microbiol. Rev. 24, 85-106. https://doi.org/10.1111/j.1574-6976.2000.tb00534.x
  8. Ennahar, S., Sonomoto, K., and Ishizaki, A. (1999) Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation. J. Biosci. Bioeng. 87, 705-716. https://doi.org/10.1016/S1389-1723(99)80142-X
  9. Gonzalez, B., Arca, P., Mayo, B., and Suarez, J. E. (1994) Detection, purification and partial characterization of plantaricin C, a bacteriocin produced by Lactobacillus plantarum strain of dairy origin. Appl. Environ. Microbiol. 60, 2158-2163.
  10. Green, G., Dicks, L. M. T., Bruggeman, G., Vandamme, E. J., and Chikindas, M. L. (1997) Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832. J. Appl. Microbiol. 83, 127-132. https://doi.org/10.1046/j.1365-2672.1997.00241.x
  11. Hoover, D. G. and Harlander, S. K. (1993) Screening methods for detecting bacteriocin activity. In: Bacteriocins of lactic acid bacteria. Hoover, D. G. and Steenson, L. R. (eds) Academic Press, NY, pp. 23-39.
  12. Jack, R. W., Tagg, J. R., and Ray. B. (1995) Bacteriocins of Gram-positive bacteria. Microbiol. Rev. 59, 171-200.
  13. Kawai, Y., Sato, T., Toba, T., Samant, S. K., and Itoh, T. (1994) Isolation and characterization of a highly hydrophobic new bacteriocin (Gassericin A) from Lactobacillus gasseri LA39. Biosci. Biotech. Biochem. 58, 1218-1221. https://doi.org/10.1271/bbb.58.1218
  14. Kim, D. S. (2002) Characteristics of the bacteriocin from Lactobacillus sp. Oh-B3. Korean J. Microbiol. Biotechnol. 30, 181-188.
  15. Kim, W. J. (1993) Bacteriocins of lactic acid bacteria: Their potentials as food biopreservative. Food Rev. Int. 9, 299-313. https://doi.org/10.1080/87559129309540961
  16. Klaenhammer, T. R. (1993) Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12, 39-85. https://doi.org/10.1111/j.1574-6976.1993.tb00012.x
  17. Kwon, D. Y. (1992) Synthesis of stereoisomeric trifluoroethylmandelates and their stereospecificity for the uses as the substrate of lipases in organic solvent. J. Korean Chem. Soc. 36, 125-129.
  18. Kwon, D. Y., Koo, M. S., Ryoo, C. R., Kang, C., H., Min, K. H., and Kim, W. J. (2002) Bacteriocin produced by Pediococcus sp. in kimchi and its characteristics. J. Microbiol. Biotechnol. 12, 96-105.
  19. Liu, W. and Hansen, J. N. (1990) Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis. Appl. Environ. Microbiol. 56, 2551-2558.
  20. Moon, G. S., Kim, W. J., and Kim, M. H. (2002) Synergistic effects of bacteriocin-producing Pediococcus acidilactici K10 and organic acids on inhibiting Escherichia coli O157:H7 and applications in ground beef. J. Microbiol. Biotechnol. 12, 936-942.
  21. Nes, I. F. and Johnsborg, O. (2004) Exploration of antimicrobial potential in LAB by genomics. Curr. Opin. Biotechnol. 15, 100-104. https://doi.org/10.1016/j.copbio.2004.02.001
  22. Oh, S. (2001) Characteristics of class II bacteriocins produced by lactic acid bacteria. J. Korean Dairy Technol. Sci. 19, 133-146.
  23. Oh, S., Kim, M. H., Churey, J. J., and Worobo, R. W. (2003) Purification and Characterization of an antilisterial bacteriocin produced by Leuconostoc sp. W65. J. Microbiol. Biotechnol. 13, 680-686.
  24. Oh, S., Roh, H., Ko, H. J., Kim, S., Kim, K. H., Lee, S. E., Chang, I. S., Kim, S. H., and Choi, I. G. (2011) Complete genome sequencing of Lactobacillus acidophilus 30SC isolated from swine intestine. J. Bacteriol. 10, 1128-1133
  25. Ray, B. (1992) Nisin of Lactococcus lactis ssp. lactis as a food biopreservative. In: Food biopreservatives of microbial origin. Ray, B. and Daeschel, M. (eds) CRC Press, Boca Raton, FL, pp. 207.
  26. Rheem, S., Oh, S., Han, K. S., Imm, J. Y., and Kim, S. H. (2002) New response surface approach to optimize medium composition for production of bacteriocin by Lactobacillus acidophilus ATCC 4356. J. Microbiol. Biotechnol. 12, 449-496.
  27. Schagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of protein in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379. https://doi.org/10.1016/0003-2697(87)90587-2
  28. Schved, F., Lalazar, Y., Henis, Y., and Juven, B. J. (1993) Purification, partial characterization and plasmid-linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacteriol. 74, 67-77. https://doi.org/10.1111/j.1365-2672.1993.tb02998.x
  29. Tagg, J. R., Dajani, A. S., and Wannamaker, L. W. (1976) Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40, 722-756.
  30. Toba, T., Samant, S. K., Yoshioka, E., and Itoh, T. (1991) Reutericin 6, a new bactriocin produced by Lactobacillus reuteri LA 6. Lett. Appl. Microbiol. 13, 281-286. https://doi.org/10.1111/j.1472-765X.1991.tb00629.x
  31. Worobo, R. W., van Belkum, M. J., Sailer, M., Roy, K. L., Vederas, J. C., and Stiles, M. E. (1995) A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens. J. Bacteriol. 177, 3143-3149.

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