Biochemical Characterization of an Extracellular Protease in Serratia proteamaculans Isolated from a Spider

무당거미에서 분리한 Serratia proteamaculans에서 분비되는 단백질분해효소의 생화학적 특성

  • Lee Kieun (Department of Biology, Chungnam National University) ;
  • Kim Chul-Hee (Department of Biology, Chungnam National University) ;
  • Kwon Hyun-Jung (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kwak Jangyul (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Shin Dong-Ha (Insect Biotech Co., Ltd.) ;
  • Park Doo-Sang (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Bae Kyung-Sook (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park Ho-Yong (Insect Resources Laboratory, Korea Research Institute of Bioscience and Biotechnology)
  • 이기은 (충남대학교 생물학과) ;
  • 김철희 (충남대학교 생물학과) ;
  • 권현정 (한국생명공학연구원 곤충자원연구실) ;
  • 곽장열 (한국생명공학연구원 곤충자원연구실) ;
  • 신동하 ((주)인섹트바이오텍) ;
  • 박두상 (한국생명공학연구원 곤충자원연구실) ;
  • 배경숙 (한국생명공학연구원 곤충자원연구실) ;
  • 박호용 (한국생명공학연구원 곤충자원연구실)
  • Published : 2004.12.01

Abstract

Serratia proteamaculans isolated from the midgut of a spider formed big halos around the bacterial colonies, indicating that the bacterial strain produces an extracellular protease. Activity staining of the extracellular pro­tein fractions using zymogram also demonstrated that the major protein with an estimated molecular mass of 52 kDa contained a high proteolytic activity. The protease was purified to near electrophoretic homogeneity from the culture supernatant after filtration and ion exchange and size exclusion chromatography. The purified enzyme had a relatively high proteolytic activity between pH 6.0 and 10.0 and at broad temperature range. The proteolytic activity of the enzyme was not inhibited by phenylmethylsulfonyl fluoride but strongly inhibited by 1, 10-phenanthroline and EDTA. The activity also was dependent on the presence of $Ca^{++}\;and\;Zn^{++}$ ions. These observations indicate that the enzyme is a metalloprotease.

Keywords

chromatography;metalloprotease;proteolytic activity;Serratia proteamaculans;zymogram

References

  1. Binet, R., S., Letoffe, J.M. Ghigo, P. Delepelaire, and C. Wandersman. 1997. Protein secretion by Gram-negative bacterial ABC exporters-a review. Gene 192, 7-11
  2. Bowen, D., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. ffrench-Constant. 1998. Insecticidal toxins from the bacterium Photorhabdus luminescens. Science 280, 2129-2132
  3. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
  4. Chabeaud, P., A. de Groot, W. Bitter, J. Tommassen, T. Heulin, and W. Achouak. 2001. Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum. J. Bacteriol. 183, 2117- 2120
  5. Claeson, G., J. Fareed, C. Larsson, G. Kindel, S. Arielly, R. Simonsson, H.L. Messmore, J.U. Balis. 1979. Inhibition of the contractile action of bradykinin on isolated smooth muscle preparations by derivatives odflow molecular weight peptides. Adv. Exp. Med. Biol. 120, 691-713
  6. Duong, F., A. Lazdunski, B. Cami, and M. Murgier. 1992. Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa : relationships to other secret ory pathways. Gene 121, 47-54
  7. Fernandez, L., P. Secades, J.R. Lopez, I. Marquez, and J.A. Guijarro. 2002. Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulence. Microbiology 148, 2233-2243
  8. Fernandez, L., J.R. Lopez, P. Secades, A. Menendez, I. Marquez, and J.A. Guijarro. 2003. In vitro and in vivo studies of the Yrp1 protease from Yersinia ruckeri and its role in protective immunity against enteric red mouth disease of salmonids. Appl. Environ. Microbiol. 69, 7328-7335
  9. Hauck, G. 1976. Proceedings: Vitalmicroscopic investigations of the effects of thrombin, a snake venom enzyme and histamin effect on the mesenteric microvasculature of rabbit and cat. Arzneimittelforschu ng 26, 1233
  10. Kawai, E., H. Akatsuka, A. Idei, T. Shibatani, and K. Omori. 1998. Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system. Mol. Microbiol. 27, 941-952
  11. Kaya, H.K. and R. Gaugler. 1993. Entomopathogenic nematodes. Annu. Rev. Entomol. 38, 181-206
  12. Liao, C.H. and D.E. McCallus. 1998. Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091. Appl. Environ. Microbiol. 64, 914-921
  13. Mackman, N., J,M. Nicaud, V. Gray, and I.B. Holland. 1986. Secretion of hemolysin by Escherichia coli. Curr. Top. Microbiol. Immunol. 125, 159-181
  14. Marty, K.B., C.L. Williams, L.J. Guynn, M.J. Benedik, and S.R. Blanke. 2002. Characterization of a cytotoxic factor in culture filtrates of Serratia marcescens. Infect. Immun. 70, 1121-1128
  15. Nakahama, K., K. Yoshimura, R. Marumoto, M. Kikuchi, I. S. Lee, T. Hase, and H. Matsubara. 1986. Cloning and sequencing of Serratia protease gene. Nucleic. Acids. Res. 14, 5843-5855
  16. Akatsura, H., E. Kawai, K. Omori, and T. Shibatani. 1995. The three genes lipB, lipC, lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide. J. Bacteriol. 177, 6381-6389
  17. Akhurst, R. 1993. Bacterial symbionts of entomopathogenic nematodes- the power behind the throne. In Nematodes and the Biological control of Insect Pests, ed. R. Bedding, R. Akhurst, H. Kaya, pp. 127-135. Melbourne, Aust
  18. Letoffe, S., P. Delepelaire, and C. Wandersman. 1991. Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J. Bacteriol. 173, 2160-2166
  19. Marits, R., V. Koiv, E. Laasik, and A. Mae. 1999. Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity. Microbiology 145, 1959-1966
  20. Sandstrom, J.P., J.A. Russell, J.P. White, and N.A. Moran. 2001. Independent origins and horizontal transfer of bacterial symbionts of aphids. Mol. Ecol. 10, 217-228
  21. Christianson, D.W. and J.D. Cox. 1999. Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes. Annu. Rev. Biochem. 68, 33-57
  22. Walshaw, D.L. and P.S. Poole. 1996. The general L-amino acid permease of Rhizobium leguminosarum is an ABC uptake system that also influences efflus of solutes. Mol. Microbiol. 21, 1239- 1252
  23. Guzzo, J., J.M. Pages, F. Duong, A. Lazdunski, and M. Murgier. 1991. Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism. J. Bacteriol. 173, 5290-5297
  24. Wolz, R.L. and J.S. 1990. Phe(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase merprin. Anal. Biochem. 191, 314-320 https://doi.org/10.1016/0003-2697(90)90225-X
  25. Guzzo, J., M. Murgier, A. Filloux, and A. Lazdunski. 1990. Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli. J. Bacteriol. 172, 942-8
  26. Kawai, E., A. Idei, H. Kumura, K. Shimazaki, H. Akatsuka, and K. Omori. 1999. The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33. Biochim. Biophys. Acta. 1446, 377-382
  27. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
  28. Braunagel, S.C. and M.J. Benedik. 1990. The metalloprotease gene of Serratia marcescens strain SM6. Mol. Gen. Genet. 222, 446-451
  29. Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, P. A2.2. Cold Spring Harbor Laboratory. Cold Spring Harbor. New York
  30. Bowen, D.J., T.A. Rocheleau, C.K. Grutzmacher, L. Meslet, M. Valens, D. Marble, A. Dowling, R. ffrench-Constant, and M.A. Blight. 2003. Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus. Microbiology 149, 1581-1591 https://doi.org/10.1099/mic.0.26171-0
  31. Braun, V. and G. Schmitz. 1980. Excretion of a protease by Serratia marcescens. Arch. Microbiol. 124, 55-61
  32. Duong, F., C. Soscia, A. Lazdunski, and M. Murgier. 1994. The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system. Mol. Microbiol. 11, 1117-1126
  33. Guzzo, J., F. Duong, C. Wandersman, M. Murgier, and A. Lazdunski. 1991. The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli $\alpha$-haemolysin. Mol. Microbiol. 5, 447-453 https://doi.org/10.1111/j.1365-2958.1991.tb02128.x
  34. Schmitz, G. and V. Braun. 1985. Cell-bound and secreted proteases of Serratia marcescens. J. Bacteriol. 161, 1002-1009
  35. Fath, M.J. and R. Kolter. 1993. ABC transporters; bacterial exporters. Microbiol. Rev. 57, 995-1017
  36. Letoffe, S., P. Delepelaire, and C. Wandersman. 1990. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli $\alpha$-haemolysin. EMBO. J. 9, 1375-1382
  37. Grkovic, S., T.R. Glare, T.A. Jackson, and G.E. Corbett. 1995. Genes essential for amber disease in grass grubs are located on the large plasmid found in Serratia entomopila and Serratia proteamaculans. Appl. Environ. Microbiol. 61, 2218-2223
  38. Moon, E.Y., H.Y. Oh, P.J. Maeng, and K.-S. Bae. 2001. Identification of enteric bacteria from Nephila clavata. Kor. J. Microbiol. 37, 1-8
  39. Caldas, C., A. Cherqui, A. Pereira, and N. Simoes. 2002. Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression. Appl. Environ. Microbiol. 68, 1297-1304
  40. Hines, D.A., P.N. Saurugger, G.M. Ihler, and M.J. Benedik. 1988. Genetic analysis of extracellular proteins of Serratia marcescens. J. Bacteriol. 170, 4141-4146