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Characterization of Protease Produced by Elizabethkingia meningoseptica CS2-1 and Optimization of Cultural Conditions for Amino Acid Production

닭 우모 분해세균 Elizabethkingia meningoseptica CS2-1이 생산하는 단백질분해효소의 특성 및 아미노산 생산을 위한 배양조건

  • 김세종 (목원대학교 미생물나노소재학과) ;
  • 조천휘 ((주)카프코 생물화학연구소) ;
  • 황경숙 (목원대학교 미생물나노소재학과)
  • Received : 2011.01.26
  • Accepted : 2011.03.22
  • Published : 2011.06.30

Abstract

A feather-degrading bacterium Elizabethkingia meningoseptica CS2-1 was isolated from compost in a chicken farm. Cultured on a basal medium containing 2% chicken feather, the bacterium showed 729.7 ${\mu}mol/mL$ of amino acid. Optimal culture conditions for feather degradation by E. meningoseptica CS2-1 were $25^{\circ}C$, pH 7.5, and 180 rpm. The optimal pH and temperature for protease activity were 8.0 and $40^{\circ}C$, respectively. The composition of an optimal medium for amino acid production was 0.05% NH4Cl, 0.05% NaCl, 0.03% $K_2HPO_4$, 0.03% $KH_2PO_4$, 0.01% $MgCl_2{\cdot}6H_2O$, 0.1% urea, and 2% chicken feather. Characteristics of amino acids extracted from the optimal medium under the optimal culture conditions of E. meningoseptica CS2-1 were analyzed. The total amino acid content of strain CS2-1 was 1063 ${\mu}mol/mL$, which was 46% higher compared to the basal condition (729.7 ${\mu}mol/mL$). The essential amino acid content in the total amino acid was 315.9 ${\mu}mol/mL$, which was 44% higher than that of the basal condition. Major amino acids were proline (14%), aspartic acid (12%), glutamic acid (11%), serine (10%), alanine (10%), glycine (9%), and tyrosine (7%) by strain CS2-1. These results suggest that strain CS2-1 can be used as a potential microbial resource for the production of amino acid using chicken feathers.

우모분으로부터 높은 함량의 아미노산을 생성하는 Elizabethkingia meningoseptica CS2-1 (729.7 ${\mu}mol/mL$)를 선발하였다. 선발 균주의 protease 활성과 우모 분해능을 위한 최적배지 및 배양조건을 확립하고 우모 분해산물 아미노산의 특성을 조사하였다. E. meningoseptica CS2-1의 최적 배양조건은 $25^{\circ}C$, pH 7.5, 180 rpm이었으며, 효소는 pH 8.0, $40^{\circ}C$에서 가장 높은 활성을 나타내었다. E. meningoseptica CS2-1의 아미노산 생산을 위한 최적 배지조건은 $NH_4Cl$ 0.05%, NaCl 0.05%, $K_2HPO_4$ 0.03%, $KH_2PO_4$ 0.03%, $MgCl_2{\cdot}6H_2O$ 0.01%, urea 0.1%, 닭 우모분 2%이었다. E. meningoseptica CS2-1를 최적 배지 및 배양조건에서 배양한 결과, 아미노산의 총 함량은 1063 ${\mu}mol/mL$로 기본조건 (729.7 ${\mu}mol/mL$)보다 46%로 향상되었고 필수 아미노산 함유량은 315.9 ${\mu}mol/mL$로 기본조건 (219.3 ${\mu}mol/mL$)보다 44%로 향상되었다. 닭 우모분으로부터 추출되 는 17 종류의 아미노산 중 proline (14%), aspartic acid (12%), serine (10%), alanine (10%), glycine (9%), tyrosine (7%)이 주요 아미노산으로 추출되는 특징을 나타내었다. 따라서, E. meningoseptica CS2-1은 닭 우모로부터 아미노산 생산을 위한 미생물유전자원 소재로서 잠재적 가치가 클 것으로 판단된다.

Keywords

References

  1. Anson (1938) The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 22, 79-89. https://doi.org/10.1085/jgp.22.1.79
  2. Asahi M, Lindquist R, Fukuyama K, Apodaca G, Epstein WL, and Mckerrow JH (1992) Purification and characterization of major extracellular proteinases from Trichophyton rubrum. Biochem J 232, 139-144.
  3. Bernal C, Cairo J, and Coello N (2005) Purification and characterization of a novel exocellular keratinase from Kocuria rosea. Enz Microb Technol 36, 211-216.
  4. Bertsch A and Coello N (2005) A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Biores Technol 96, 1703-1708. https://doi.org/10.1016/j.biortech.2004.12.026
  5. Bockle B, Galunski B, and Muller R (1995) Characterization of a keratinolytic serine protease from Streptomyces pactum DSM40530. Appl Environ Microbiol 61, 3705-3710.
  6. Brysk MM and Rajaraman S (1992) Cohesion and desquamation of epidermal stratum corneum. Prog Histochem Cytochem 25, 1-53.
  7. Chitte RR, Nalawade VK, and Dey S (1999) Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus SDS. Lett Appl Microbiol 28, 131-136. https://doi.org/10.1046/j.1365-2672.1999.00484.x
  8. Chon DH, Kang SM, and Kwon TJ (2003) Purification and some properties of protease produced by Pseudomonas sp. KP-364. Kor J Microbiol Biotechnol 31, 224-229.
  9. Chu IM, Lee C, and Li TS (1992) Production and degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC14416. Enz Microb Technol 14, 755-761. https://doi.org/10.1016/0141-0229(92)90116-6
  10. Gupta R and Ramnani P (2006) Microbial keratinase and their prospective application: An overview. Appl Microbiol Biotechnol 70, 21-33. https://doi.org/10.1007/s00253-005-0239-8
  11. Hong SJ, Namkung H, Kim WY, and Paik IK (2002) Effects of supplemental feather digests on the growth of broiler chicks and taurine content in the broiler meat. Kor J Poult Sci 29, 141-147.
  12. Hood CM and Healy MG (1994) Bioconversion of waste keratins: Wool and feathers. Conserv Recyc 11, 179-188. https://doi.org/10.1016/0921-3449(94)90088-4
  13. Kim JH and Ko YD (2005) Effect of dietary protease (bromelain) treated feather meal on the performance and nutrient utilization in broilers. J Anim Sci Technol 47, 221-232. https://doi.org/10.5187/JAST.2005.47.2.221
  14. Kim SJ, Cho CH, and Whang KS (2010) Isolation and characterization of keratinolytic protein chicken feather-degrading bacteria. Kor J Microbiol 46, 86-92.
  15. Lee KH, Park KK, Park SH, and Lee JB (1987) Isolation, purification and characterization of keratinolytic proteinase from Microsporum canis. Yonsei Med J 28, 131-138. https://doi.org/10.3349/ymj.1987.28.2.131
  16. Lin X, Inglis GD, Yanke LJ, and Cheng KJ (1999) Selection and characterization of feather-degrading bacteria from canola meal compost. J Ind Microbiol Biotechol 23, 149-153. https://doi.org/10.1038/sj.jim.2900706
  17. Lin X, Lee CG, Casale ES, and Shih JCH (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl Environ Microbiol 58, 3271-3275.
  18. Lobarzewski J, Grzywnowicz K, Wawrzkiewicz K, Staszczak M, and Wolski T (1990) Feather keratin as a ligand in an affinity chromatographic technique for isolation of protease from Trichophyton verrucosum. J Chromat 520, 223-235. https://doi.org/10.1016/0021-9673(90)85106-6
  19. Mitsuiki S, Ichikawa M, Oka T, Sakai M, Moriyama Y, Sameshima Y, Goto M, and Furukawa K (2004) Molecular characterization of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1. Enz Microb Technol 34, 482-489. https://doi.org/10.1016/j.enzmictec.2003.12.011
  20. Nagal S and Jain PC (2010a) Production of feather hydrolysate by Elizabethkingia meningoseptica KB042 (MTCC 8360) in submerged fermentation. Indian J Microbiol 50, 41-45. https://doi.org/10.1007/s12088-010-0014-0
  21. Nagal S, Kango N, and Jain PC (2010b) Production of alkaline protease from Elizabethkingia meningoseptica KB042 using chicken feathers. Ann Microbiol 60, 629-635. https://doi.org/10.1007/s13213-010-0101-9
  22. Onifade AA, Al-Sane NA, Al-Musallam AA, and Al-Zarban S (1998) A review: Potentials for biotechnological applications of keratin-degrading 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
  23. Parag AM and Hassan MA (2004) Purification, characterization and immobilization of a keratinase from Aspergillus oryzae. Enz Microb Technol 34, 85-93. https://doi.org/10.1016/j.enzmictec.2003.09.002
  24. Riffel A, Brandelli A, Bellato CM, Souza GHMF, Eberlin MN, and Tavares FCA (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
  25. Sangali S and Brandelli A (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. Singh CJ (2002) Optimization of an extracellular protease of Chrysosporium keratinophilum and its potential in bioremediation of keratinic wastes. Mycophathologia 156, 151-156.
  27. Takami H, Nakamura S, Aono R, and Horikoshi K (1992) Degradation of human hair by a thermostable alkaline protease from alkalophilic Bacillus sp. No. AH 101. Biosci Biotech Biochem 56, 1667-1669. https://doi.org/10.1271/bbb.56.1667
  28. Tsuboi R, Ko I, Takamori K, and Ogawa H (1989) Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH. Infect Immun 57, 3479-3483.
  29. Vignardet C, Guillaume YC, Friedrich J, and Millet J (1999) A first order experimental design to assess soluble proteins released by a new keratinase from Doratomyces microsporus on human substrate. Int J Pharm 191, 95-102. https://doi.org/10.1016/S0378-5173(99)00283-5
  30. Wang JJ and Shih JCH (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 EO, Kim MJ, Ryu EY, Park GT, Lee CY, Son HJ, and Lee SJ (2007a) Isolation and Application of Feather-Degrading Bacteria for Development of Environment-Friendly Biofertilizer. J Environ Sci 16, 1103-1109. https://doi.org/10.5322/JES.2007.16.9.1103
  32. Woo EO, Kim MJ, Son HS, Ryu EY, Jeong SY, Son HJ, Lee SJ, and Park GT (2007b) Production of 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
  33. Yamamura S, Morita Y, Hasan Q, Rao SR, Murakami Y, Yokoyama K, and Tamiya E (2002) Characterization of a new keratin-degrading bacterium isolated from deer fur. J Biosci Bioeng 93, 595-600. https://doi.org/10.1016/S1389-1723(02)80243-2
  34. Zaghloul TI, Al-Bahra M, and Al-Azmeh H (1998) Isolation, identification, and keratinolytic activity of several feather-degrading bacterial isolates. Appl Biochem Biotechnol 70, 207-213. https://doi.org/10.1007/BF02920137