Journal of Life Science (생명과학회지)

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

DOI QR Code

Purification and Characterization of the Staphylococcus epidermidis Urease

Staphylococcus epidermidis urease의 정제 및 생화학적 특성에 관한 연구

  • 민선희 (대구가톨릭대학교 약학대학) ;
  • 이만형 (대구가톨릭대학교 약학대학)
  • Published : 2007.04.25
Download PDF
⟨ Previous Next ⟩

Abstract

Staphylococcus epidermidis is a coagulase-negative, gram-positive bacterium that normally inhabits the human skin. S. epidermidis is also known to be an opportunistic pathogen in infections of various indwelling medical devices. This report describes purification and characterization of the urease of S. epidermidis urease, which may act as a virulence factor. The urease from S. epidermidis was purified 1,127 fold by using DEAE-Sepharose, Phenyl-Sepharose, Mono-Q and Superdex HR200 column chromatography. The specific activity of the purified enzyme was 993.8 U/mg. Michaelis constant($K_m$) of the enzyme was estimated to be 8.5 mM urea by using Lineweaver-Burke double reciprocal plot. The native molecular weight of the urease was shown to be 255 kD by using Superose 6HR gel filtration chromatography and the purified enzyme contained 2.2 nickel ions per catalytic unit. The overall stoichiometry of the enzyme subunits appears to be $(\alpha\beta\gamma)_3$, which is consistent with the enzymes from other bacteria sources.

본 연구에서는 피부상재균이며 기회병원균이기도 한 Staphylococcus epidermidis ATCC12228로부터 urease효소를 4단계 크로마토그라피 방법을 사용하여 1,127배 정제하고 그 생화학적인 특성을 규명하였다. 정제된 urease 효소는 SDS-PACE 전기영동분석 및 gel-filtration 크로마토그라피를 이용한 천연분자량 분석결과, 67, 16.1 및 12.7 kDa의 3개 subunit가 3량체로 회합되어 존재하는 것으로 나타났으며 catalytic unit 당 2.2개의 니켈 원소를 함유하는 것으로 측정되었다. 정제된 효소의 비활성은 993.8 U/mg, $K_m$값은 8.5mM로 각각 산출되었다.

Keywords

References

  1. Archer, G. L., G. J. Vazquez and J. L. Johnston. 1980. Antibiotic prophylaxis of experimental endocarditis due to methicillin-resistant Staphylococcus epidermidis. J. Infect. Dis. 142, 725-731 https://doi.org/10.1093/infdis/142.5.725
  2. Baddour, L. M., T. N. Phillips and A. L. Bisno, 1986. Coagulase-negative staphylococcal endocarditis. Occurrence in patients with mitral valve prolapse. Arch. Intern. Med. 146, 119-121 https://doi.org/10.1001/archinte.146.1.119
  3. Benini, S., W. R. Rypniewski, K. S. Wilson, S. Miletti, S. Ciurli and S. Mangani. 1999. A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels. Structure. 7, 205-216 https://doi.org/10.1016/S0969-2126(99)80026-4
  4. Dunne, W. M., Jr. 2002. Bacterial adhesion: seen any good biofilms lately? Clin. Microbiol. Rev. 15, 155-166 https://doi.org/10.1128/CMR.15.2.155-166.2002
  5. Gatermann, S., J. John and R. Marre. 1989. Staphylococcus saprophyticus urease: characterization and contribution to uropathogenicity in unobstructed urinary tract infection of rats. Infect. Immun. 57, 110-116
  6. Gatermann, S. and R. Marre. 1989. Cloning and expression of Staphylococcus saprophyticus urease gene sequences in Staphylococcus carnosus and contribution of the enzyme to virulence. Infect. Immun. 57, 2998-3002
  7. Gill, S. R., D. E. Fouts, G. L. Archer, E. F. Mongodin, R. T. Deboy, J. Ravel, I. T. Paulsen, J. F. Kolonay, L. Brinkac, M. Beanan, R. J. Dodson, S. C. Daugherty, R. Madupu, S. V. Angiuoli, A. S. Durkin, D. H. Haft, J. Vamathevan, H. Khouri, T. Utterback, C. Lee, G. Dimitrov, L. Jiang, H. Qin, J. Weidman, K. Iran, K. Kang, I. R. Hance, K. E. Nelson and C. M. Fraser. 2005. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J. Bacteriol. 187, 2426-2438 https://doi.org/10.1128/JB.187.7.2426-2438.2005
  8. Goldmann, D. A. and G. B. Pier. 1993. Pathogenesis of infections related to intravascular catheterization. Clin. Microbiol. Rev. 6, 176-192 https://doi.org/10.1128/CMR.6.2.176
  9. Gruter, L., M. Endres and S. Gatermann. 1992. Cloning and expression of Staphylococcus epidermidis urease gene sequences in Staphylococcus carnosus. FEMS Microbiol. Lett. 72, 33-35
  10. Huebner, J. and D. A. Goldmann. 1999. Coagulase-negative staphylococci: role as pathogens. Annu. Rev. Med. 50, 223-236 https://doi.org/10.1146/annurev.med.50.1.223
  11. Jabri, E., M. B. Carr, R. P. Hausinger and P. A. Karplus. 1995. The crystal structure of urease from Klebsiella aerogenes. Science 268, 998-1004 https://doi.org/10.1126/science.7754395
  12. Kloos, W. E. and T. L. Bannerman. 1994. Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev. 7, 117-140 https://doi.org/10.1128/CMR.7.1.117
  13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  14. Leighton, P. M. and J. A. Little. 1986. Identification of coagulase-negative Staphylococci isolated from urinary tract infections. Am. J. Clin. Pathol. 85, 92-95 https://doi.org/10.1093/ajcp/85.1.92
  15. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275
  16. Martin, M. A., M. A. Pfaller and R. P. Wenzel. 1989. Coagulase-negative staphylococcal bacteremia. Mortality and hospital stay. Ann. Intern. Med. 110, 9-16 https://doi.org/10.7326/0003-4819-110-1-9
  17. Mobley, H. L. and R. P. Hausinger. 1989. Microbial ureases: significance, regulation, and molecular characterization. Microbiol. Rev. 53, 85-108
  18. Mobley, H. L., M. D. Island and R. P. Hausinger. 1995. Molecular biology of microbial ureases. Microbiol. Rev. 59, 451-480
  19. O'Gara, J. P. and H. Humphreys. 2001. Staphylococcus epidermidis biofilms: importance and implications. J. Med. Microbiol. 50, 582-587 https://doi.org/10.1099/0022-1317-50-7-582
  20. Rubin, J., W. A. Rogers, H. M. Taylor, E. D. Everett, B. F. Prowant, L. V. Fruto and K. D. Nolph. 1980. Peritonitis during continuous ambulatory peritoneal dialysis. Ann. Intern. Med. 92, 7-13 https://doi.org/10.7326/0003-4819-92-1-7
  21. Schafer, U. K. and H. Kaltwasser. 1994. Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease. Arch. Microbiol. 161, 393-399
  22. Schierholz, J. M. and J. Beuth. 2001. Implant infections: a haven for opportunistic bacteria. J. Hosp. Infect. 49, 87-93 https://doi.org/10.1053/jhin.2001.1052
  23. Tacconelli, E., M. Tumbarello, M. Pittiruti, F. Leone, M. B. Lucia, R. Cauda and L. Ortona. 1997. Central venous catheter-related sepsis in a cohort of 366 hospitalised patients. Eur. J. Clin. Microbiol. Infect. Dis. 16, 203-209 https://doi.org/10.1007/BF01709582
  24. Vuong, C. and M. Otto. 2002. Staphylococcus epidermidis infections. Microbes. Infect. 4, 481-489 https://doi.org/10.1016/S1286-4579(02)01563-0
  25. Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971-974 https://doi.org/10.1021/ac60252a045
  26. Zhang, Y. Q., S. X. Ren, H. L. Li, Y. X. Wang, G. Fu, J. Yang, Z. Q. Qin, Y. G. Miao, W. Y. Wang, R. S. Chen, Y. Shen, Z. Chen, Z. H. Yuan, G. P. Zhao, D. Qu, A. Danchin and Y. M. Wen. 2003. Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Mol. Microbiol. 49, 1577-1593 https://doi.org/10.1046/j.1365-2958.2003.03671.x

Cited by

  1. Characterization of Urease-Producing Bacteria Isolated from Heavy Metal Contaminated Mine Soil vol.47, pp.6, 2014, https://doi.org/10.7745/KJSSF.2014.47.6.391