난분해성 케라틴 단백질을 함유하는 닭 우모 분해세균의 분리 및 특성

Isolation and Characterization of Keratinolytic Protein Chicken Feather-Degrading Bacteria

  • 김세종 (목원대학교 미생물나노소재학과) ;
  • 조천휘 ((주)카프코 생물화학연구소) ;
  • 황경숙 (목원대학교 미생물나노소재학과)
  • 투고 : 2010.02.25
  • 심사 : 2010.03.15
  • 발행 : 2010.03.31

초록

양계장 부산물 시료로부터 우모 분해세균 31균주를 분리하였다. 수집된 우모 분해세균에 대해 16S rRNA 염기서열을 해석하여 계통학적 특성을 검토한 결과, Firmicutes (21균주), ${\gamma}$-proteobacteria (4균주), Actinobacteria (4균주) 그리고 Bacteroidetes (2균주) 계통군에 속하는 다양한 세균이 확보되었다. 우모 분해세균 중 우모 분해율이 75-90% 이상인 우수균주 Chryseobacterium sp. FBF-7, Stenotrophomonas maltophilia FBS-4 그리고 Lysinibacillus sp. FBW-3를 최종 선발하였다. 이들 균주를 이용한 생물학적 분해법과 Ca(OH)2를 이용한 화학적 분해법에 의해 추출된 아미노산의 특성을 비교한 결과, Chryseobacterium sp. FBF-7에 의해 추출된 총 아미노산 함량이 1661.6 ${\mu}mol$/ml로 선발 균주 중 가장 높게 나타났으며, 화학적 분해법에 의해 추출된 총 아미노산 함량과 유사하였다. Chryseobacterium sp. FBF-7에 의해 생성된 필수 아미노산 함유량은 619.3 ${\mu}mol$/ml로 총 아미노산의 37%를 함유하였으며, 화학적 분해법에 의한 경우, 596.9 ${\mu}mol$/ml의 필수 아미노산을 생산하여 총 아미노산 함유량의 32%를 차지하는 것으로 나타났다. Chryseobacterium sp. FBF-7에 의해 추출된 아미노산의 조성을 검토한 결과, valine, glutamic acid, aspartic acid, glycine 그리고 proline이 주요 아미노산이었으며, 특히 aspartic acid, threonine, serine, cysteine 그리고 tyrosine이 화학적 추출법보다 더 높게 추출되는 특징을 나타내었다.

Thirty-one chicken feather-degrading bacteria were isolated from wasted feather, compost and wastewater in a chicken farm. These isolates were categorized as Firmicutes (21 strains), ${\gamma}$-proteobacteria (4 strains), Actinobacteria (4 strains), and Bacteroidetes (2 strains) by 16S rRNA gene sequence analysis. We examined the feather-degrading isolates for degradation in the 2% of chicken feather meal. The strain Chryseobacterium sp. FBF-7, Stenotrophomonas maltophilia FBS-4, and Lysinibacillus sp. FBW-3 were selected as a keratinolytic protein degrading bacteria which showed the highest feather degradation of 75-90%. The characteristics of amino acids extracted from chicken feather meal by using keratinolytic protein degrading isolates and chemical method with $Ca(OH)_2$ were analyzed. Total amino acid content of strain Chryseobacterium sp. FBF-7 was 1,661.6 ${\mu}mol$/ml, which was the highest and it was similar with chemical method. And essential amino acid content of total amino acid was thirty-seven percent (619.3 ${\mu}mol$/ml) and 596.9 ${\mu}mol$/ml for keratinolytic protein degrading isolates and chemical method, respectively. The major amino acids were valine, glutamic acid, aspartic acid, glycine, and proline by the strain Chryseobacterium sp. FBF-7 and especially, higher contents of aspartic acid, threonine, serine, cysteine, and tyrosine were detected compared with chemical method.

키워드

참고문헌

  1. Ahmed, I., A. Yokota, A. Yamazoe, and T. Fujiwara. 2007. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int. J. Syst. Bacteriol. 57, 1117-1125. https://doi.org/10.1099/ijs.0.63867-0
  2. Baker, D.H., R.C. Blitenthal, K.P. Boebel, G.L. Czarnecki, L.L. Southern, and G.M. Willis. 1981. Protein-amino acid evalluation of steam-processed feather meal. Poult. Sci. 60, 1865-1872. https://doi.org/10.3382/ps.0601865
  3. Bockle, B., B. Galunski, and R. Muller. 1995. Characterization of a keratinolytic serine protease from Streptomyces pactum DSM40530. Appl. Environ. Microbiol. 61, 3705-3710.
  4. Chon, D.H., S.M. Kang, and T.J. Kwon. 2003. Purification and some properties of keratinolytic protease produced by Pseudomonas sp. KP-364. Kor. J. Microbiol. Biotechnol. 31, 224-229.
  5. Colette, M.H. and G.H. Michael. 1994. Bioconversion of waste keratins: wool and feathers. Conserv. Recyc. 11, 179-188. https://doi.org/10.1016/0921-3449(94)90088-4
  6. Gupta, R. and P. Ramnani. 2006. Microbial keratinase and their prospective application: an overview. Appl. Microbiol. Biotechnol. 70, 21-33. https://doi.org/10.1007/s00253-005-0239-8
  7. Herzog. P., I. Winkler, D. Wolking, P. Kampfer, and A. Lipski. 2008. Chryseobacterium ureilyticum sp. nov., Chryseobacterium gambrini sp. nov., Chryseobacterium pallidum sp. nov. and Chryseobacterium molle sp. nov., isolated from beer-bottling plants. Int. J. Syst. Evol. Microbiol. 58, 26-33. https://doi.org/10.1099/ijs.0.65362-0
  8. Kim, J.M., W.J. Lim, and H.J. Suh. 2001. Feather-degrading Bacillus species from poultry waste. Process Biochem. 37, 287- 291. https://doi.org/10.1016/S0032-9592(01)00206-0
  9. Kim, W.K. and P.H. Patterson. 2000. Nutritional value of enzymeor sodium hydroxide treated feathers from dead hens. Poult. Sci. 79, 528-534. https://doi.org/10.1093/ps/79.4.528
  10. Kim, Y.B., J.B. Lee, K.S. Sung, and N.H. Lee. 1998. Effects of physical processing on protein content and pepsin digestibility of feather meals. Kor. J. Anim. Sci. 40, 103-110.
  11. Korea Chicken Council. 2002. www.chicken.or.kr/chicken/data/ state.htm.
  12. Lee, N.H., Y.B. Kim, H.J. Kim, K.S. Seong, J.H. Rho, and C.K. Han. 1999. Effects of physiochemical treatment on the isolation of keratinaceous protein and amino acids of feather meal. Kor. J. Anim. Nutr. Feed. 23, 15-20.
  13. Lin, X., C.C. Lee, E.S. Casale, and J.C.H. Shih. 1992. Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl. Environ. Microbiol. 58, 3271- 3275.
  14. Marchisio, V.F., A. Fusconi, and S. Rigo. 1994. Keratinolysis and its morphological expression in hair digestion by airborne fungi. Mycopathologia 127, 103-115. https://doi.org/10.1007/BF01103066
  15. Moore, E.R.B., A.S. Kruger, L. Hauben, S.E. Seal, R. De Baere, R. De Wachter, K.N. Timmis, and J. Swings. 1997. 16S rRNA gene sequence analysis and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia. FEMS Microbiol. Lett. 151, 145-153. https://doi.org/10.1111/j.1574-6968.1997.tb12563.x
  16. Mukhopadyay, R.P. and A.L. Chandra. 1990. Keratinase of Streptomycete. Indian J. Exp. Bio. 28, 575-577.
  17. Oyeka, C.A. and H.C. Gugnani. 1997. Keratin degradation by Scytalidium species and Fusarium solani. Mycoses 41, 73-76.
  18. Papadopoulose, M.C. 1985. Processed chicken feathers as feedstuff for poultry and swine. A review. Agric. Wastes 14, 275-290. https://doi.org/10.1016/S0141-4607(85)80009-3
  19. Papadopoulose, M.C., A.R. El Boushy, and A.E. Roodbeen. 1985. The effect of varying autoclaving conditions and added sodium hydroxide on amino acid content and nitrogen characteristics of feather meal. J. Sci. Food Agric. Abstr. 36, 1219-1226. https://doi.org/10.1002/jsfa.2740361204
  20. Qin, L.M., S. Dekio, and J. Jidoi. 1992. Some biochemical characteristics of a partially purified extacellular keratinase from Trichophyton schoenleinii. Zentralbl. Bakteriol. 277, 236-244. https://doi.org/10.1016/S0934-8840(11)80618-3
  21. Onifade, A.A., N.A. Al-Sane, A.A. Al-Musallam, and A. Al- Zarban. 1998. A review: Potentials for biotechnological applications of keratin-de-grading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Biores. Technol. 66, 1-11. https://doi.org/10.1016/S0960-8524(98)00033-9
  22. Riffel, A. and A. Brandelli. 2000. Isolation and characterization of a feather-degrading bacterium from the poultry processing industry. J. Ind. Microbiol. Biotechnol. 29, 255-258.
  23. Riffel, A., A. Brandelli, C.M. Bellato, G.H.M.F. Souza, M.N. Eberlin, and F.C.A. Tavares. 2007. Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J. Biotechnol. 128, 693-703. https://doi.org/10.1016/j.jbiotec.2006.11.007
  24. Saitou, N. and M. Nei. 1987. The neighbor-joinning method: a new method for reconstruction phylogenetic trees. Mol. Biol. Evol. 4, 406-426.
  25. Sangali, S. and A. Brandelli. 2000. Feather keratin hydrolysis by a Vibrio sp. strain kr2. J. Appl. Microbiol. 89, 735-743. https://doi.org/10.1046/j.1365-2672.2000.01173.x
  26. Santos, R.M.D.B., A.A.P. Firmino, C.M de Sa, and C.R Felix. 1996. Keratinolytic activity of Aspergillus fumigatus. Curr. Microbiol. 33, 364-370. https://doi.org/10.1007/s002849900129
  27. Son, H.J., G.T. Park, and Y.G. Kim. 2004. Production of a keratinolytic protease by a feather-degrading bacterium, Bacillus megaterium F7-1. Kor. J. Microbiol. 40, 43-48.
  28. Taha, I.Z. 1998. Cloned Bacillus subtilis alkaline protease(apr A) gene showing high level of keratinolytic activity. Appl. Biochem. Biotechnol. 70/72, 199-205 https://doi.org/10.1007/BF02920136
  29. 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
  30. Wang, J.J. and J.C.H. Shih. 1999. Fermentation production of keratinase from Bacillus licheniformis PWD-1 and a recombinant B. subtilis FDB-29. J. Ind. Microbiol. Biotechnol. 22, 608-616. https://doi.org/10.1038/sj.jim.2900667
  31. Woo. E.O., M.J. Kim, H.S. Son, E.Y. Ryu, S.Y. Jeong, H.J. Son, S.J. Lee, and G.T. Park. 2007. Production of keratinolytic protease by Bacillus pumilus RS7 and feather hydrolysate as a source of amino acids. J. Environ. Sci. 16, 1203-1208. https://doi.org/10.5322/JES.2007.16.10.1203