생명과학회지 (Journal of Life Science)

  • 제23권2호
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  • Pages.273-281
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  • 2013
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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해수에서 분리한 Micrococcus sp. PS-1이 생산하는 단백질 분해효소의 생산과 효소학적 특성

Production and Characterization of Alkaline Protease of Micrococcus sp. PS-1 Isolated from Seawater

  • 진영랑 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 유선녕 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김광연 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김상헌 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 박슬기 (부산대학교 의학전문대학원 미생물학 및 면역학 교실) ;
  • 김현경 (부산대학교 의학연구원) ;
  • 이용석 (동아대학교 생명과학자원대학 생물공학과) ;
  • 최용락 (동아대학교 생명과학자원대학 생물공학과) ;
  • 지재훈 (아주대학교 의과대학 유전체불안정제어 연구센터) ;
  • 안순철 (부산대학교 의학전문대학원 미생물학 및 면역학 교실)
  • Jin, Young-Rang (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Yu, Sun-Nyoung (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Kwang-Youn (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Sang-Hun (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Park, Seul-Ki (Department of Microbiology and Immunology, Pusan National University School of Medicine) ;
  • Kim, Hyeun-Kyeung (Medical Research Institute, Pusan National University) ;
  • Lee, Yong-Seok (Department of Biotechnology, Dong-A University) ;
  • Choi, Yong-Lark (Department of Biotechnology, Dong-A University) ;
  • Ji, Jae Hoon (Genome Instability Research Center, Ajou University School of Medicine) ;
  • Ahn, Soon-Cheol (Department of Microbiology and Immunology, Pusan National University School of Medicine)
  • 투고 : 2013.01.18
  • 심사 : 2013.02.20
  • 발행 : 2013.02.28
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초록

본 연구는 부산 인근의 해수욕장에서 얻은 해수에서 protease를 생산하는 균주를 분리하여 동정하고 균주의 배양학적인 특성과 protease의 효소학적 특성을 확인하였다. 해수에서 분리한 protease를 생산하는 미생물은 16S rDNA sequencing을 통해 Micrococcus sp. PS-1으로 동정하였다. Protease 생산의 최적조건은 2% skim milk와 1% NaCl이 포함된 pH 7.0의 LB배지에 48시간 배양이었다. 효소의 부분정제를 위해 ultrafiltration과 acetone 침전법을 사용하였고, zymography를 통해 분자량이 35.0 kDa과 37.5 kDa인 protease를 확인하였다. 또한 효소의 최적 활성은 pH 9.0와 $37^{\circ}C$에서 나타났고, 효소는 pH 8.0에서 11.0까지, $25^{\circ}C$에서 $37^{\circ}C$까지 80% 이상의 효소활성이 유지되어 안정한 것으로 확인되었으며 PMSF, EDTA 처리시 protease가 저해되는 것을 통해 alkaline metallo-serine protease로 확인되었다.

The purpose of this research was to investigate the production and characterization of alkaline protease from Micrococcus sp. PS-1 newly isolated from seawater. Micrococcus sp. PS-1 was grown in Luria-Bertani (LB) medium. Its optimal temperature and pH for growth were $30^{\circ}C$ and 7.0, respectively. The effect of nitrogen sources was investigated on optimal enzyme production. A high level of alkaline protease production occurred in LB broth containing 2% skimmed milk. The protease was purified in a 3-step procedure involving ultrafiltration, acetone precipitation, and dialysis. The procedure yielded a 16.43-purification fold, with a yield of 54.25%. SDS-PAGE showed that the enzyme had molecular weights of 35.0 and 37.5 kDa. Its maximum protease activity was exhibited at pH 9.0 and $37^{\circ}C$, and its activity was stable at pH 8.0-11.0 and $25-37^{\circ}C$. The protease activity was strongly inhibited by PMSF, EDTA, and EGTA. Taken together, the results demonstrate that the protease enzyme from Micrococcus sp. PS-1 probably belongs to a subclass of alkaline metallo-serine proteases.

키워드

참고문헌

  1. Amoozegar, M. A., Fatemi, A. Z., Karbalaei-Heidarib, H. R. and Razavi, M. R. 2007. Production of an extracellular alkaline metallo protease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiol Res 4, 369-377.
  2. Arulmani, M., Aparanjini, K., Vasanthi, K., Arumugam, P., Arivuchelvi, M. and Kalaichelvan, T. 2007. Purification and partial characterization of serine protease from thermostable alkalophilic Bacillus laterosporus-AK1. World J Microbiol Biotechnol 23, 475-481. https://doi.org/10.1007/s11274-006-9249-7
  3. Cavicchioli, R., Siddiqui, K. S., Andrews, D. and Sowers, K. R. 2002. Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13, 253-261. https://doi.org/10.1016/S0958-1669(02)00317-8
  4. Clark, D. J., Hawrylik, S. J., Kavanagh, E. and Opheim, D. J. 2000. Purification and characterization of a unique alkaline elastase from Micrococcus luteus. Protein Expr Purif 18, 46-55. https://doi.org/10.1006/prep.1999.1166
  5. Choi, N. S., Choi, J. H., Kim, B. H., Han, Y. J., Kim, J. S., Lee, S. G. and Song, J. J. 2009. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 12, 2234-2237.
  6. Choi, N. S., Hahm, J. H., Maeng, P. J. and Kim, S. H. 2005. Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, ${\beta}$-mercaptoethanol, DTT, SDS, Triton X-100, and Urea. J Biochem Mol Biol 38, 177-181. https://doi.org/10.5483/BMBRep.2005.38.2.177
  7. Choi, N. S., Kim, B. Y., Lee, J. Y., Yoon, K. S., K. Han, Y. and Kim, S. H. 2002. Relationship between acrylamide concentration and enzymatic activity in an improved single fibrin zymogram gel system. J Biochem Mol Biol 35, 236-238. https://doi.org/10.5483/BMBRep.2002.35.2.236
  8. Cristobal, H. A., Schmidt, A., Kothe, E., Breccia, J. and Abate, C. M. 2009. Characterization of inducible cold-active ${\beta}$-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-antarctic ecosystem. Enzyme Microb Technol 45, 498-506. https://doi.org/10.1016/j.enzmictec.2009.06.010
  9. Cristobal, H. A., Breccia, J. D. and Abate, C. M. 2008. Isolation and molecular characterization of Shewanella sp. G5, a producer of cold-active ${\beta}$-D-glucosidases. J Basic Microbiol 48, 16-25. https://doi.org/10.1002/jobm.200700146
  10. Fernandez, J., Mohedano, A. F., Polanco, M. J., Medina, M. and Nunez, M. 1996. Purification and characterization of an extracellular cysteine proteinase produced by Micrococcus sp. INIA 528. J Appl Bacteriol 81, 27-34. https://doi.org/10.1111/j.1365-2672.1996.tb03278.x
  11. Hartley, B. S. 1960. Proteolytic enzymes. Annu Rev Biochem 29, 45-72. https://doi.org/10.1146/annurev.bi.29.070160.000401
  12. Jonathan, K., Burkhardt, F., Stephen, A. J., David, P. H. L., John, P. M., Fergal, O. G., and Alan, D. W. D. 2010. Marine metagenomics: New tools for the study and exploitation of marine microbial metabolism. Mar Drugs 8, 608-628. https://doi.org/10.3390/md8030608
  13. Joo, H. S., Kumar, C. G., Park, G. C., Paik, S. R. and Chang, C. S. 2003. Oxidant and SDS-stable alkaline protease from Bacillus clausiiI-52: production and some properties. J Appl Microbiol 95, 267-272. https://doi.org/10.1046/j.1365-2672.2003.01982.x
  14. Kunitz, M. 1947. Crystalline soyabean trypsin inhibitor II. general properties. J Gen Physiol 30, 291-310. https://doi.org/10.1085/jgp.30.4.291
  15. Lee, J. H., Shin, H. H., Lee, D. S., Kwon, K. K., Kim, S. J., and Lim, H. K. 1999. Bacterial diversity of cultivable isolates from sea water and a marine coral, Plexauridae sp. near Mun-Sum, Cheju-Island. J Microbiol 37, 193-199.
  16. Li, H. Zheng, P., H., Yuan, J. L., Fan, H. D., Chen, W., Wang, S. J., Zheng, S. S., Zheng, Z. L. and Zou, G. L. 2007. A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization. Phytother Res 12, 1234-1241.
  17. Mala, B. R., Aparna, M. T., Mohini, S. G. and Vasanti, V. D. 1998. Molecular and biotechnological aspect of microbial protease. Microbiol Mol Biol 62, 597-635.
  18. Massaki, Y., Kazuo, S. and Mitsuo, M. 1984. Purification and properties of acid protease from Monascus sp. No. 3403. Agric Biol Chem 48, 1637-1639. https://doi.org/10.1271/bbb1961.48.1637
  19. Mohsen, F. N. 2005. Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron J Biotechnol 8, 197-203. https://doi.org/10.2225/vol8-issue2-fulltext-5
  20. Nicholas, C. P. and Lewis, S. 1999. Fundamentals of Enzymology, pp. 35-36, 3rd eds., Oxford University press, UK.
  21. Park, P. J., Lee, S. H., Byun, H. G., Kim, S. H. and Kim, S. K. 2002. Purification and characterization of a collagenase from the mackerel, Scomber japonicus. J Biochem Mol Biol 35, 576-582. https://doi.org/10.5483/BMBRep.2002.35.6.576
  22. Prasad, R., Malik, R. K. and Mathur, D. K. 1986. Purification and characterization of extracellular caseinolytic enzyme of Micrococcus Sp. MCC-315 isolated from Cheddar cheese. J Dairy Sci 69, 633-642. https://doi.org/10.3168/jds.S0022-0302(86)80450-7
  23. Rajesh, P., Mital, D. and Satya, P. S. 2005. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochem 40, 3569-3575. https://doi.org/10.1016/j.procbio.2005.03.049
  24. Reis, V. R. and Zydney, A. 2001. Membrane separations in biotechnology. Curr Opin Biotechnol 12, 208-211. https://doi.org/10.1016/S0958-1669(00)00201-9
  25. Runying, Z., Rui, Z., Jing Z. and Nianwei, L. 2003. Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization. Extremophiles 7, 335-337. https://doi.org/10.1007/s00792-003-0323-x
  26. Tarun, B. and Elmer, H. M. 1989. Purification and partial characterization of an aminopeptidase from Micrococcus freudenreichii ATCC 407 systematic and applied microbiology. Syst Appl Microbiol 12, 112-118. https://doi.org/10.1016/S0723-2020(89)80046-3

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