Purification and Characterization of Two Extracellular Proteases from Oligotropha carboxydovorans DSM 1227


초록

Two extracellular proteases, EP I and EP II, from cells of Oligotropha carboxydovorans (formerly Pseudomonas carboxydovorans) DSM 1227 grown in nutrient broth were purified to greater than 95% homogeneity in five steps using azocasein as a substrate. The final specific activities of EPs I and II were 214.9 and 667.4 units per mg of protein. The molecular weights of native EPs I and II were determined to be 23,000. Sodium dodecyl sulfate-gel electrophoresis revealed the two enzymes to be monomers. The enzymes were found to be serine-type proteases. The activity of EP I was stimulated by Ca2+, Mg2+, and Ba2+, but that of EP II was not. The enzymes were completely inhibited by Fe2+, Hg2+, Co2+, Zn2+, and Cd2+. EDTA and EGTA exhibited a strong inhibitory effect on EP I. The optimal pH for the two enzymes was pH 9.0. The optimal temperatures for EP I and II were 60 and 50$^{\circ}C$, respectively. The enzymes were stable under alkaline conditions. The thermal stability of EP I was higher than that of EP II. Cell-free extracts did not inhibit the purified enzymes. The enzymes were active on casein, azocasein, azocoll, and carbon monoxide dehydrogenase, but weakly active with bovine serum albumin.

키워드

참고문헌

  1. Biochem. J. v.91 Estimation of the molecular weights of proteins by Sephadex gel filtration Andrews, P.
  2. Kor. J. Microbiol. v.30 Purification and characterization of an intracellular protease from Pseudomonas carboxydovorans DSM 1227 grown on carbon monoxide Bae, K.H.;Y.M. Kim
  3. Annu. Rev. Biochem. v.56 Intracellular proteases Bond, J.S.;P.E. Butler
  4. Can. J. Microbiol. v.25 Regulation of extracellular protease formation by Serratia marcescens Bromke, B.J.;J.M. Hammel
  5. Biochem. Biophys. Acta v.391 Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa Carrick, L.;R.S. Berk
  6. Eur. J. Biochem. v.146 Mechanisms of activation and secretion of a cell-associated precursor of an exocellular protease of Pseudomonas aeruginosa 34362A Fecycz, I.T.;J.N. Campbell
  7. Annu. Rev. Biochem. v.45 Intracellular protein degradation in mammalian and bacterial cells Goldberg, A.L.;A.C. St. John
  8. Nature v.228 In vivo degradation of nonsense fragments in Escherichia coli Goldschmidt, R.
  9. J. Gen. Microbiol. v.99 Characterization of Serratia marcescens, S. liquefactens, S. plymuthica and S. marinorubra by electrophoresis of their proteinases Grimont, P.A.D.;F. Grimont;H.L.C. Dulong
  10. Kor. J. Microbiol. v.27 Extracellular proteases from Bacillus licheniformis: partial purification and characterization Hong, N.;M. Choi;C. Yang
  11. J. Bacteriol. v.155 Purification and properties of serine protease from Halobacterium halobium Izotova, L.S.;A.Y. Strongn;L.N. Chekulaeva;V.E. Sterkin;V.I. Ostoslavskaya;L.A. Lyublinskaya;E.A. Timokhina;V.M. Stepanov
  12. J. Bacteriol. v.144 Nutritional factors controlling exocellular protease production by Pseudomonas aeruginosa Jenson, S.E.;I.T. Fecycz;J.N. Campbell
  13. Can. J. Microbiol. v.26 Purification and characterization of exocellular proteases produced by clinical isolate and a laboratory strain of Pseudomonas aeruginosa Jensen, S.E.;L. Phillippe;J.T. Tseng;G.W. Stremke;J.N. Campbell
  14. Kor. Biochem. J. v.23 Purification and some properties of two intracellular proteases from Acinetobacter sp. strain JC1 DSM 3803 Kim, E.-B.;Y.M. Kim
  15. J. Bacteriol. v.171 Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803 Kim, K.S.;Y.T. Ro;Y.M. Kim
  16. J. Bacteriol. v.148 Purification and some properties of carbon monoxide dehydrogenase from Pseudomonas carboxydohydrogena Kim, Y.M.;G.D. Hegeman
  17. Intl. Rev. Cytol. v.81 Oxidation of carbon monoxide by bacteria Kim, Y.M.;G.D. Hegeman
  18. Infec. Immun. v.19 Purification of Pseudomonas aeruginosa protease and microscopic characterization of pseudomonal protease induced rabbit coroneal damage Kreger, A.S.;L.D. Gray
  19. J. Bacteriol. v.151 Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis Kurotsu, T.;M.A. Marahiel;K.-D. Muller;H. Kleinkauf
  20. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli, U.K.
  21. Kor. J. Microbiol. v.29 Purification and characterization of an intracellular protease from Pseudomonas carboxydohydrogena Lee, H.S.;Y.M. Kim
  22. Kor. J. Microbiol. v.27 Purification and characterization of an intracellular protease from Pseudomonas carboxydovorans Lee, J.H.;Y.M. Kim
  23. J. Biol. Chem. v.244 Bacterial peptidases Levy, C.C.;P. Goldman
  24. Biol. Chem. v.193 Protein measurement with the folin phenol reagent wry, O.H.;N.J. Rosebrough;A.L. Farr;R.J. Randall
  25. Mol. Microbiol. v.3 Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi Letoffe, S.;P. Delepelaire;C. Wandersman
  26. Eur. J. Biochem. v.171 Characterization of a 73 KD thiol protease from Serratia marcescens and its effect on plasma proteins Mella, A.;Y. Yamamoto;H. Maeda
  27. Autotrophic bacteria Aerobic carbon monoxide-oxidizing bacteria Meyer, O.;H.G. Schlegel(ed.);B. Bowien(ed.)
  28. System. Appl. Microbiol. v.16 Reclassification of ubiquinone Q-10-containing carboxidotrophic bacteria: Transfer of "[Pseudomonas] carboxydovorans" OM5¹ to Oligotropha, gen.nov., as Oligotropha carboxydovorans, comb.nov., transfer of "[Alcaligenes] carboxydus" $DSM1086^T$ to Cabophilus, gen.nov. as Carbophilus carboxydus, comb.nov., transfer of "[Pseudomonas] compransoris" $DSM1231^T$ to Zavarzinia, gen.nov, as Zavarzinia compransoris, comb.nov., and amended descriptions of the new genera Meyer, O.;E. Stackebrandt;G. Auling
  29. Arch. Microbiol. v.118 Reisolation of the carbon monoxide-utilizing hydrogen bacterium Pseudomonas carboxydovorans (Kistner) com.nov Meyer, O.;H.G. Schlegel
  30. Adv. Enzymol. v.41 Comparative specificity of microbial proteinases Morihara, K.
  31. Annu. Rev. Microbiol. v.118 Turnover of intracellular proteins Pine, M.J.
  32. J. Bacteriol. v.170 Control of intracellular serine protease expression in Bacillus subtilis Ruppen, M.E.;G.L.V. Alstine;L. Band
  33. J. Bacteriol. v.153 Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease Sastry, K.J.;O.P. Srivastva;J.Millet;P.C. Fitz.James;A.I. Aronson
  34. J. Bacteriol. v.159 Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein Vasantha, N.;L.D. Thompson;C. Rhodes;C. Banner;J. Nayle;D. Filpula
  35. J. Bacteriol. v.131 Purification of the extracellular protease of Bacillus licheniformis and its inhibition by bacitracin Vitkovic, L.;H.L. Sadoff
  36. J. Bacteriol. v.169 Characterization of Erwinia crysanthemi extracellular proteases: Cloning and expression of the protease genes in Escherichia coli Wandersman, C.;P. Delepelaire;S. Letoffe;M. Schwartz
  37. J. Gen. Microbiol. v.132 Extracellular proteases in Erwinia crysanthemi Wandersman, C.;T. Andro;Y. Bertheau
  38. J. Biol. Chem. v.244 The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis Weber, K.;M. Osborn
  39. J. Gen. Microbiol. v.103 Purification and properties of protease with elastase activity from Pseudomonas aeruginosa Wretlind, B.;T. Wadstrom