Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Screening and Identification of Alkaline Protease Inhibitor-Producing Marine-derived Actinomyces.

Alkaline protease inhibitor를 생산하는 해양유래 방선균의 탐색 및 동정

  • Kang, Sung-Il (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Kong, Jai-Yul (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Choi, Yeung-Joon (Division of Marine Bioscience/Institute of Marine Industry, Gyeongsang National University) ;
  • Kim, Min-Yong (Department of Refrigeration Engineering, Chonnam National University) ;
  • Son, Hong-Joo (School of Applied Life Science, Pusan National University)
  • 강성일 (부경대학교 생물공학과) ;
  • 공재열 (부경대학교 생물공학과) ;
  • 최영준 (경상대학교 해양식품생명공학과) ;
  • 김민용 (전남대학교 냉동공학과) ;
  • 손홍주 (부산대학교 생명응용과학부)
  • Published : 2008.04.30
Download PDF
⟨ Previous Next ⟩

Abstract

In this study, we screened and identified the bacterial strain showing high alkaline pretense inhibitor activity from marine environment. Nine bacterial strains with alkaline pretense inhibitor activity were isolated from marine sediments. Among them, strain C12 had the highest alkaline pretense inhibitor activity and was selected for further taxonomical study. On the basis of morphological and physiological characteristics, strain C12 was identified as the genus Streptomyces. A phylogenetic analysis of the 165 rDNA showed that the isolated strain was actually a member of the genus Streptomyces, because the determined sequence exhibited a higher homology with Streptomyces thermocarboxydus. Morphological characteristics showed cylindrical spore chain and smooth spore surface by scanning electron microscope. Strain C12 was grown on all media except for ISP 9 agar. This strain could be grown in the medium containing up to 9% NaCl.

Alkaline protease는 식품 가공 공정에 있어 가공 원료의 선도 저하를 초래하거나 맛살류 제품 원료인 어류의 근육조직을 파괴하여 gel 구조를 파괴시키는 것으로 알려져 있다. 본 연구는 해양으로부터 alkaline protease에 대한 저해력을 가지는 방선균들을 분리하여 그 중 저해력이 가장 높은 C12균주를 최종 선정하였다. 본 균주의 형태학적, 배양적 및 생리학적 특성을 조사한 결과, 포자의 크기는 $2.0\;{\mu}m$로 외형은 원통형이고, 편모가 없으며, 포자형태는 smooth하였다. ISP 9배지를 제외한 대부분의 배지에서 잘 성장하였다. 또한 $15{\sim}50^{\circ}C$에서 잘 성장하였으며, 9% (w/v) NaCl이 포함된 배지에서도 성장하는 것으로 확인되었다. Gram 양성, citrate 음성, catalase 양성이었으며, melanin 색소를 생성하지 않았다. Starch, casein 및 gelatin 분해능이 있었으며, glucose, galactose, maltose, lactose, fructose 및 mannse 등은 잘 이용하였지만, sorbitol과 sucrose는 이용하지 않았다. 이러한 특성을 토대로 본 균주는 Streptomyces sp.로 확인되었다. 보다 정확한 균주 동정을 위하여 16S rDNA 염기서열 분석을 수행하였으며, 그 결과 C12 균주는 S. thermocarboxydus와 계통진화학적으로 가장 유연관계가 높았다.

Keywords

References

  1. An, H., M. Y. Peters and T. A. Seymour. 1996. Roles of endogenous enzymes in surini gelation. J. Food Sci. 7, 321-326
  2. Barrett, A. J. and G. Salvesen. 1986. Proteinase inhibitors. pp. 3-18, Elsevier Science Publishers B. V
  3. Bode, W. and R. Huber. 1992. Natural protein proteinase inhibitors and their interaction with proteinases. Eur. J. Biochem. 204, 433-451 https://doi.org/10.1111/j.1432-1033.1992.tb16654.x
  4. Demuth, H. U. 1990. Recent developments in inhibiting cysteine and serine proteases. J. Enzym. Inhib. 3, 249-278 https://doi.org/10.3109/14756369009030375
  5. Miyairi, K., A. Ogasawara, A. Tonouchi, K. Hosaka, M. Kudou and T. Okuno. 2004. Low-molecular-weight pectate lyase from Streptomcyces thermocarboxydus. J. Appl. Glycosci. 51, 1-7 https://doi.org/10.5458/jag.51.1
  6. Kato, J., S. Hirano, Y. Ohnishi and S. Horinouchi. 2005. The Streptomyces subtilisin inhibitor (SSI) gene in Streptomyces coelicolor A3 (2). Biosci. Biotechnol. Biochem. 69, 1624-1629 https://doi.org/10.1271/bbb.69.1624
  7. Kenney, A. J. 1999. Nomenclature and classes of peptidase, pp. 1-8. In Sterchi, E. E. and W. Stocker (eds.), Proteolytic Enzyme, Springer Lab Manual, New York
  8. Kreig, N. R and J. G. Holt. 1989. Bergey's Manual of Systematic Bacteriology. pp. 2451-2492, Williams ＆ Wilkins. Baltimore, Maryland
  9. Kuramoto, A., A. Lezhava, S. Taguchi, H. Momose and H. Kinashi. 1996. The location and deletion of the genes which code for SSI-like protease inhibitors in Streptomyces species. FEMS Microbiol. Lett. 139, 37-42 https://doi.org/10.1111/j.1574-6968.1996.tb08176.x
  10. Moore, S. and W. H. Stein. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176, 367-388
  11. Orr, G. L., J. A. Strickland and T. A. Walsh. 1994. Inhibition of diabrotica larval growth by a multicystatin from potato tubers. J. Insect Physiol. 40, 893-900 https://doi.org/10.1016/0022-1910(94)90023-X
  12. Pandhare, J., K. Zog and V. V. Deshpande. 2002. Differential stability of alkaline protease inhibitors from actinomyces: effect of various additives on thermostability. Biores. Technol. 84, 165-169 https://doi.org/10.1016/S0960-8524(02)00025-1
  13. Ryan, C. A. 1990. Protease inhibitors in plants : Genes for improving defense against insects and pathogens. Annu. Rev. Phytopathol. 28, 25-49
  14. Saeki, H., Z. Iseya, S. Sugiura and N. Seki. 1995. Gel forming characteristics of frozen surimi from Chum salmon in the presence inhibitors. J. Food Sci. 60, 917-922 https://doi.org/10.1111/j.1365-2621.1995.tb06261.x
  15. Saitou, N. and M. Nei. 1987. The neighbor-joining method : a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  16. Terabe, M., S. Kojima, S. Taguchi, H. Momose and K. Miura. 1994. Primary structure and inhibitory properties of a subtilisin-chymotrypsin inhibitor from Streptomyces virginiae. Eur. J. Biochem. 226, 627-632 https://doi.org/10.1111/j.1432-1033.1994.tb20089.x
  17. Terashita, T., M. Kono and S. Murao. 1980. Promoting effect of S-PI on fruiting of Lentinus edodes. Trans. Mycol. Soc. Jpn. 21, 137-140
  18. Travis, J. and G. S. Salvesen. 1983. Human plasma proteinase inhibitors. Ann. Rev. Biochem. 52, 655-709 https://doi.org/10.1146/annurev.bi.52.070183.003255
  19. Umezawa, H. 1972. Enzyme inhibitors of microbial origin, pp. 1-114, University of Tokyo Press, Tokyo
  20. Ustadi, K., K. Y. Kim and S. M. Kim. 2005. Characteristics of protease inhibitor purified from the eggs of Alaska pollock (Theragra chalcogramma). J. Kor. Fish. Soc. 38, 83-88 https://doi.org/10.5657/kfas.2005.38.2.083
  21. Vernekar, J. V., A. M. Tanksale, M. S. Ghatge and V. V. Deshpande. 2001. Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity. Biochem. Biophys. Res. Commun. 285, 1018-1024 https://doi.org/10.1006/bbrc.2001.5257
  22. Weerasinghe, V. C., M. T. Morrissey and H. An. 1996. Characterization of active components in food grade protinase inhibitor for surimi manufacture. J. Agric. Food Chem. 44, 2584-2590 https://doi.org/10.1021/jf950589z
  23. Yamashita, M. and S. Konagaya. 1991. Cysteine protease inhibitor in egg of Chum salmon. J. Biochem. 110, 762-766 https://doi.org/10.1093/oxfordjournals.jbchem.a123655

Cited by

  1. A Study on the Correlation between Odorous Compounds, Actinomycetes and Algae in Drinking Water Source of Nakdong River vol.35, pp.3, 2013, https://doi.org/10.4491/KSEE.2013.35.3.213