Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Purification and Characterization of β-Lactamase Secreted from Bacillus sp. J105 Strain having β-Lectam Antibiotics Resistance.

(β-lactam계 항생물질 저항성을 지닌 Bacillus sp. J105 균주로부터 분비되는 베타 락탐 분해효소의 정제 및 특성

  • Cho, Kyeong-Soon (Public Health and Environment Institute of Pusan) ;
  • Kang, Byoung-Won (BK21 Center for Silver-Bio industrialization Project, Dong-A University) ;
  • Seo, Min-Jeong (Department of Biotechnology, Dong-A University) ;
  • Lee, Young-Choon (Department of Biotechnology, Dong-A University) ;
  • Lee, Jai-Heon (Department of Genetic Engineering, Dong-A University) ;
  • Joo, Woo-Hong (Department of Biology, Changwon National University) ;
  • Choi, Yung-Hyun (Department of Biochemistry, College of Oriental Medicine, Dong-eui University) ;
  • Lim, Hak-Seob (Department Bioinstitute, MILLENNIUM PROMISE CO., LTD.) ;
  • Kim, Jeong-In (School of Food and Life Science, Inje University) ;
  • Seo, Kwon-Il (Department of Food and Nutrition, Sunchon National University) ;
  • Jeong, Yong-Kee (Department of Biotechnology, Dong-A University, Department Bioinstitute, MILLENNIUM PROMISE CO., LTD.)
  • 조경순 (부산광역시 보건환경연구원) ;
  • 강병원 (동아대학교 BK21 실버바이오 사업단) ;
  • 서민정 (동아대학교 생명공학과) ;
  • 이영춘 (동아대학교 생명공학과) ;
  • 이재헌 (동아대학교 유전공학과) ;
  • 주우홍 (창원대학교 생물학과) ;
  • 최영현 (동의대학교 한의학대학 생화학교실) ;
  • 임학섭 ((주)천년약속 바이오연구소) ;
  • 김정인 (인제대학교 식품생명과학부) ;
  • 서권일 (순천대학교 식품영양학과) ;
  • 정영기 (동아대학교 생명공학과, (주)천년약속 바이오연구소)
  • Published : 2008.06.30
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Abstract

${\beta}-Lactamase$, secreted from Bacillus sp. J105 strain was purified to a single band on SDS-PAGE by ammonium sulfate precipitation, ion exchange column chromatography and gel-filtration. The molecular weight of the purified enzyme was 31 kDa on SDS-PAGE and its isoelectric point was 7.35. Optimal pH and temperature for enzymatic reaction were 5 and $40^{\circ}C$, respectively. As a result of total amino acid composition analysis of the purified enzyme, Gly and Ala were occupied 14.1 and 13.3 mole %, respectively. Km and Vmax value of purified enzyme were 1.33 mM and 0.36 mM/ml using ampicillin as a substrate, respectively.

Bacillus sp. J105 strain으로부터 유도된 ${\beta}-lactamase$는 ammonium sulfate 침전, 이온 교환 칼럼 크로마토그래피, 겔 여과 등의 과정을 거쳐 SDS-PAGE에서 단일 band로 정제하였다. 정제된 효소의 분자량은 31 kDa이었으며 등전점은 7.35이었다. 효소반응의 최적 pH와 온도는 각각 5와 $40^{\circ}C$이었다. 정제 단백질의 총 아미노산 조성의 분석 결과, Gly과 Ala이 각각 14.1과 13.3 mole%로 가장 많은 아미노산 잔기를 차지하고 있었다. 정제 효소의 ampicillin을 기질로 하였을 때의 Km값은 1.33 mM이었고 Vmax값은 0.36 mM/ml이었다.

Keywords

References

  1. Bush, K., G. A. Jacoby and A. A. Medeiros. 1995. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39, 1211-1233 https://doi.org/10.1128/AAC.39.6.1211
  2. Cho, K. S., W. D. Kang, D. H. Kim, H. Beak, H. K. Sung and Y. K. Joung. 1998. Characterization of resistant bacterium against $\beta$-lactam antibiotics. J. Dong-eui univ. 28, 403-410
  3. Danziger, L. H. and S. L. Pendland. 1995. Bacterial resistance to $\beta$-lactam antibiotics. Am. J. Health-Syst. Pharmacy 52, 3-8
  4. Fisher, J. F., S. O. Meroueh and S. Mobashery. 2005. Bacterial resistance to $\beta$-lactam antibiotics: Compelling opportunism, compelling opportunity. Chem. Rev. 105, 395-424 https://doi.org/10.1021/cr030102i
  5. Fu, K. P. and H. C. Neu. 1981. The role of inducible $\beta$- lactamases in the antagonisms seen with certain cephalosporin combinations. J. Antimicrob. Chemother. 7, 104-107 https://doi.org/10.1093/jac/7.1.104
  6. Ghuysen, J. M. 1991. Serine-lactamases and penicillinbinding proteins. Annu. Rev. Microbiol. 45, 37-67 https://doi.org/10.1146/annurev.mi.45.100191.000345
  7. Hashizume, T., A. Yamaguchi and T. Sawai. 1986. Outer membrane permeability of imipenem in comparison with other $\beta$-lactam antibiotics. J. Antibiot. 1, 153-156
  8. Jan, E. P., B. L. Masecar and M. J. Jervos. 1988. Characterization and comparison of two penicillinase-producing strains of Streptococcus (Enterococcus) faecalis. Antimicrob. Agents and Chemother. 32, 122-124 https://doi.org/10.1128/AAC.32.1.122
  9. Joris, B. 1988. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R6l DD-peptidase family. Biochem. J. 250, 313-324 https://doi.org/10.1042/bj2500313
  10. Kasic, J. E. and L. Peacham. 1968. Properties of $\beta$-lactamase produced by three species of Mycobacteria. Biochem. J. 107, 675 -682 https://doi.org/10.1042/bj1070675
  11. Kelly, J. A., O. Dideberg, P. Charlier, J. P. Wery, M. Libert, P. C. Moews, J. R. Knox, C. Duez, C. Fraipont and B. Joris. 1986. On the origin of bacterial resistance to penicillin: Comparison of a beta-lactamase and a penicillin target. Science 231, 1429-1431 https://doi.org/10.1126/science.3082007
  12. Kitzis, M. D., D. Billet-klein, F. W. Goldstein, R. Williamson, G. T. Van Nhieu, J. Carlot and J. F. Acar. 1988. Dissemination of the novel plasmid-mediated $\beta$-lactamase CTX-1, which confers resistance to broad-spectrum cephalosporins, and its inhibition by $\beta$-lactamase inhibitors. Antimicrob. Agents and Chemother. 32, 9-14 https://doi.org/10.1128/AAC.32.1.9
  13. Kushner, D. J. and C. Broil. 1977. Penicillinase formation by blue green algae. Microbiology 112, 219-223 https://doi.org/10.1099/00221287-112-2-219
  14. 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
  15. Lampe, M. F., J. B. Allan, B. H. Minshew and J. C. Sherris. 1982. Mutational enzymatic resistance of Enterobacter species to beta-lactam antibiotics. Antimicrob. Agents and Chemother. 21, 655-660 https://doi.org/10.1128/AAC.21.4.655
  16. Lineweaver, H. and D. Burk. 1934. The Determination of Enzyme Dissociation Constants. J. Am. Chem. Soc. 56, 658-666 https://doi.org/10.1021/ja01318a036
  17. Livermore, D. M. 1995. beta-Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8, 557-584
  18. Livermore, D. M., F. Moosdeen, M. A. Lindridge, P. Kho and J. D. Williams. 1986. Behaviour of TEM-1 beta-lactamase as a resistance mechanism to ampicillin, mezlocillin and azlocillin in Escherichia coli. J. Antimicrob. Chemother. 17, 139-146 https://doi.org/10.1093/jac/17.2.139
  19. Lowry, O. H., N. J. Posebrough, L. A. Farr and R. J. Randal. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275
  20. Massova, I. and S. Mobashery. 1998. Kinship and diversification of bacterial penicillin-binding proteins and beta- lactamases. Antimicrob. Agents Chemother. 42, 1-17 https://doi.org/10.1093/jac/42.1.1
  21. Mehta, R. J. and C. H. Nash. 1978. $\beta$-Lactamase activity in yeast. J. Antibiot. 31, 239-240 https://doi.org/10.7164/antibiotics.31.239
  22. Minami, S., A. Yotsuji, M. Inoue and S. Mitsuhashi. 1980. Induction of $\beta$-lactamase by various b-lactam anti-biotics in Enterobacter cloacae. Antimicrob. Agents and Chemother. 18, 382-385 https://doi.org/10.1128/AAC.18.3.382
  23. Minami, S., M. Inoue and S. Mitsuhashi. 1980. Purification and properties of acephalosporinase from Enterobacter cloacae. Antimicrob. Agents and Chemother. 18, 853-857 https://doi.org/10.1128/AAC.18.6.853
  24. Neu, H. C. 1984. Current mechanisms of resistance to anti- microbial agents in micro-organisms causing infection in the patient at risk for infection. Am. J. Med. 76(6A), 11
  25. Ogawara, H. 1978. Production and property of $\beta$-lactamase in Streptomyces. Antimicrob. Agents and Chemother. 8, 402-408
  26. Poole, K. 2004. Resistance to beta-lactam antibiotics. Cell. Mol. Life Sci. 61, 2200-2223
  27. Sawai, T., I. Takahashi and S. Yamagishi. 1970. Iodometric assay method for beta-lactamase with various beta-lactam antibiotics as substrates. J. Bacteriol. 13, 910-913
  28. Sawai, T., M. Kano and K. Tsukamoto. 1982. Characterization of eight $\beta$-lactamases of Gram-negative bacteria. J. Bacteriol. 152, 567-571
  29. Weber, K. and M. Osborn. 1971. Measurement of molecular weights by electrophoresis on SDS-polyacrylamide gel. Meth. Enzymol. 26, 3-27
  30. Wu, W., P. S. F. Mezes, Y. Q. Yanag, R. W. Balacher and J. O. Lampen. 1985. Cloning and sequencing of the $\beta$-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis. J. Bacteriol. 163, 487-492