Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Aug;61(8):3098–3104. doi: 10.1128/aem.61.8.3098-3104.1995

Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri

M Klingeberg, B Galunsky, C Sjoholm, V Kasche, G Antranikian
PMCID: PMC1388561  PMID: 16535107

Abstract

The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS-resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide chain with a molecular mass of 68 kDa, showed a broad temperature and pH profile (50 to 100(deg)C; pH 5 to 11). The optimal activity with substantial thermal stability was measured with casein at 85(deg)C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and diisopropylfluorophosphate demonstrated that the enzyme was a serine proteinase. The enzyme displayed a relatively narrow substrate specificity, catalyzing the hydrolysis only of N-protected p-nitroanilides or p-nitrophenyl esters of basic (Arg or Lys) or hydrophobic (Phe or Tyr) l-amino acids. l-Phenylglycine amide was also attacked by the proteinase, but with a lower specificity constant. Within the detection limit, no hydrolysis of d-amino acid derivatives was observed. The catalytic efficiency of the enzyme at 80(deg)C (k(infcat)/K(infm) for benzoyl-Arg-p-nitroanilide, 10(sup4)) is the same order of magnitude when compared with that of functionally similar mesophilic enzymes. The proteinase also acts as a transferase, catalyzing the acyl transfer from protected amino acid ester or amide to amino acid amide. The observed thermostability, SDS resistance, relatively narrow substrate specificity, high stereospecificity, and limited catalytic efficiency probably reflect the tighter packing of the thermostable protein molecule and its limited flexibility. This was supported by fluorescence spectra of the enzyme, mainly due to tryptophan residues, in the temperature range of 30 to 90(deg)C. Structural reorganization was observed at temperatures over 100(deg)C. The results obtained could be of relevance for the better understanding of the structure-function relationship of enzymes from extreme thermophiles and suggest possible biotechnological application of the proteinase for resolution of racemic mixtures.

Full Text

The Full Text of this article is available as a PDF (305.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersson L. -O., Borg H., Mikaelsson M. Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett. 1972 Feb 1;20(2):199–202. doi: 10.1016/0014-5793(72)80793-2. [DOI] [PubMed] [Google Scholar]
  2. Ascenzi P., Menegatti E., Guarneri M., Bortolotti F., Antonini E. Catalytic properties of serine proteases. 2. Comparison between human urinary kallikrein and human urokinase, bovine beta-trypsin, bovine thrombin, and bovine alpha-chymotrypsin. Biochemistry. 1982 May 11;21(10):2483–2490. doi: 10.1021/bi00539a030. [DOI] [PubMed] [Google Scholar]
  3. Berezin I. V., Kazanskaya N. F., Klyosov A. A. Determination of the individual rate constants of alpha-chymotrypsin-catalyzed hydrolysis with the added nucleophilic agent, 1,4-butanediol. FEBS Lett. 1971 Jun 10;15(2):121–124. doi: 10.1016/0014-5793(71)80037-6. [DOI] [PubMed] [Google Scholar]
  4. Blumentals I. I., Robinson A. S., Kelly R. M. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus. Appl Environ Microbiol. 1990 Jul;56(7):1992–1998. doi: 10.1128/aem.56.7.1992-1998.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burlini N., Magnani P., Villa A., Macchi F., Tortora P., Guerritore A. A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochim Biophys Acta. 1992 Aug 21;1122(3):283–292. doi: 10.1016/0167-4838(92)90406-4. [DOI] [PubMed] [Google Scholar]
  6. Cowan D. A. Biotechnology of the Archaea. Trends Biotechnol. 1992 Sep;10(9):315–323. doi: 10.1016/0167-7799(92)90257-v. [DOI] [PubMed] [Google Scholar]
  7. Cowan D. A., Smolenski K. A., Daniel R. M., Morgan H. W. An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C. Biochem J. 1987 Oct 1;247(1):121–133. doi: 10.1042/bj2470121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. GALLY J. A., EDELMAN G. M. The effect of temperature on the fluorescence of some aromatic amino acids and proteins. Biochim Biophys Acta. 1962 Jul 16;60:499–509. doi: 10.1016/0006-3002(62)90869-7. [DOI] [PubMed] [Google Scholar]
  9. Gabel D., Kasche V. Autolysis of beta-trypsin. Influence of calcium ions and heat. Acta Chem Scand. 1973;27(6):1971–1981. doi: 10.3891/acta.chem.scand.27-1971. [DOI] [PubMed] [Google Scholar]
  10. Herbert R. A. A perspective on the biotechnological potential of extremophiles. Trends Biotechnol. 1992 Nov;10(11):395–402. doi: 10.1016/0167-7799(92)90282-z. [DOI] [PubMed] [Google Scholar]
  11. Heussen C., Dowdle E. B. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem. 1980 Feb;102(1):196–202. doi: 10.1016/0003-2697(80)90338-3. [DOI] [PubMed] [Google Scholar]
  12. JOVIN T., CHRAMBACH A., NAUGHTON M. A. AN APPARATUS FOR PREPARATIVE TEMPERATURE-REGULATED POLYACRYLAMIDE GEL ELECTROPHORESIS. Anal Biochem. 1964 Nov;9:351–369. doi: 10.1016/0003-2697(64)90192-7. [DOI] [PubMed] [Google Scholar]
  13. Kelleher P. J., Juliano R. L. Detection of proteases in polyacrylamide gels containing covalently bound substrates. Anal Biochem. 1984 Feb;136(2):470–475. doi: 10.1016/0003-2697(84)90246-x. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laderman K. A., Asada K., Uemori T., Mukai H., Taguchi Y., Kato I., Anfinsen C. B. Alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli. J Biol Chem. 1993 Nov 15;268(32):24402–24407. [PubMed] [Google Scholar]
  16. Laderman K. A., Davis B. R., Krutzsch H. C., Lewis M. S., Griko Y. V., Privalov P. L., Anfinsen C. B. The purification and characterization of an extremely thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. J Biol Chem. 1993 Nov 15;268(32):24394–24401. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lin X., Tang J. Purification, characterization, and gene cloning of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. J Biol Chem. 1990 Jan 25;265(3):1490–1495. [PubMed] [Google Scholar]
  19. Morihara K. Comparative specificity of microbial proteinases. Adv Enzymol Relat Areas Mol Biol. 1974;41(0):179–243. doi: 10.1002/9780470122860.ch5. [DOI] [PubMed] [Google Scholar]
  20. Rodbard D., Kapadia G., Chrambach A. Pore gradient electrophoresis. Anal Biochem. 1971 Mar;40(1):135–157. doi: 10.1016/0003-2697(71)90087-x. [DOI] [PubMed] [Google Scholar]
  21. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  22. Vihinen M. Relationship of protein flexibility to thermostability. Protein Eng. 1987 Dec;1(6):477–480. doi: 10.1093/protein/1.6.477. [DOI] [PubMed] [Google Scholar]
  23. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  24. Wolin E. A., Wolfe R. S., Wolin M. J. Viologen dye inhibition of methane formation by Methanobacillus omelianskii. J Bacteriol. 1964 May;87(5):993–998. doi: 10.1128/jb.87.5.993-998.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES