Advertisement

Current Microbiology

, Volume 24, Issue 2, pp 111–117 | Cite as

Isolation and characterization of an alkaline protease from the marine shipworm bacterium

  • H. L. Griffin
  • R. V. Greene
  • M. A. Cotta
Article

Abstract

Bacterial isolates from the gland of Deshayes of the marine shipworm (Psiloteredo healdi) produced extracellular protease activity when cultured with 1% cellulose. A protease with a relative molecular mass of 36,000 daltons as determined by SDS-PAGE and a pI of 8.6 was isolated from the medium and purified to electrophoretic homogeneity. No carbohydrate appeared to be associated with the protein. The enzyme was activated and stabilized by relatively high salt concentrations (>0.2M). Below 0.1M salt, significant protein aggregation occurred, as well as autohydrolysis of the protease, both of which resulted in the loss of activity. The specific activity of the enzyme was 65,840 proteolytic units/mg with azocasein substrate of optimal temperature (42°C), pH (9.0), and salt concentration (0.20M NaCl). The activity was stable up to 40°C, from pH 3.0 to pH 11.9, and from 0.1M to 3.5M NaCl. These stabilities, as well as the protease's stability in the presence of chelators, oxidizing agents, and heavy metals, suggest the enzyme has potential for use in relatively low temperature (40°C) industrial applications.

Keywords

Enzyme Cellulose Heavy Metal Molecular Mass Salt Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Austin B (1989) Novel pharmaceutical compounds from marine bacteria. J Appl Bacteriol 67:461–470PubMedGoogle Scholar
  2. 2.
    Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedGoogle Scholar
  3. 3.
    Brakke MK (1963) Photometric scanning of centrifuged density gradient columns. Anal Biochem 5:271–283PubMedGoogle Scholar
  4. 4.
    Cotta MA, Hespell RB (1986) Proteolytic activity of the ruminal bacteriumButyrivibrio fibrisolvens. Appl Environ Microbiol 52:51–58PubMedGoogle Scholar
  5. 5.
    Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determining sugars and related substances. Anal Chem 28:350–356Google Scholar
  6. 6.
    Edman P, Begg G (1967) A protein sequentor. Eur J Biochem 1:80–91PubMedGoogle Scholar
  7. 7.
    Gianazza E, Righetti PG (1979) Facts and artifacts in isoelectric focusing. In: Redola BJ (ed) Electrophoresis. Berlin, New York: Walter de, Gruyter, pp 129–140Google Scholar
  8. 8.
    Grant WD, Mwatha WE, Jones BE (1990) Alkaliphiles: ecology, diversity and applications. FEMS Microbiol Rev 75:255–270Google Scholar
  9. 9.
    Greene RV, Freer SN (1986) Growth characteristics of a novel nitrogen-fixing cellulolytic bacterium. Appl Environ Microbiol, 52:982–986Google Scholar
  10. 10.
    Greene RV, Griffin HL, Freer SN (1988) Purification and characterization of an extracellular endoglucanase from the marine shipworm bacterium. Arch Biochem Biophys 267:334–341PubMedGoogle Scholar
  11. 11.
    Greene RV, Cotta MA, Griffin HL (1990) A novel, symbiotic bacterium isolated from marine shipworm secretes proteolytic activity. Curr Microbiol 19:353–356Google Scholar
  12. 12.
    Griffin HL, Freer SN, Greene RV (1987) Extracellular endoglucanase activity by a novel bacterium isolated from marine shipworm. Biochem Biophys Res Commun 144:143–151PubMedGoogle Scholar
  13. 13.
    Hewick RM, Hunkapiller MW, Hood LE, Dryer WJ (1981) A gas-liquid solid phase peptide and protein sequentor. J Biol Chem 256:7990–7997PubMedGoogle Scholar
  14. 14.
    Kirk RE, Othmer DE (1980) Enzyme detergents and industrial enzymes. In: Kirk-Ozhzaer encyclopedia of chemical technology, 3rd edn, Vol. 9. New York: John Wiley & Sons, pp 138–224Google Scholar
  15. 15.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  16. 16.
    Martin RG, Ames BN (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem 236:1372–1379PubMedGoogle Scholar
  17. 17.
    Saravani G-A, Cowan DA, Daniel RM, Morgan HW (1989) Caldolase, a chelator-insensitive extracellular serine proteinase from aThermus spp. Biochem J 262:409–416PubMedGoogle Scholar
  18. 18.
    Scheuer PJ (1990) Some marine ecological phenomena: chemical basis and biomedical potential. Science 248:173–177PubMedGoogle Scholar
  19. 19.
    Waterbury JB, Calloway CB, Turner RD (1983) A cellulolytic nitrogen-fixing bacterium cultured from the gland of Deshayes in shipworm (Bivalvia: Teredinidae) Science 221:1401–1403Google Scholar
  20. 20.
    Waterbury JB, Calloway CB, Turner RD (1989) Bacteria for cellulose digestion. U.S. Patent Number 4,861,721Google Scholar
  21. 21.
    Whitaker JR (1972) Principles of enzymology for the food sciences. New York: Marcel Dekker, IncGoogle Scholar
  22. 22.
    Williams KW, Soderberg L (1979) A carrier ampholyte for isoelectric focusing. International Laboratory, Jan/FebGoogle Scholar
  23. 23.
    Zaborsky OR, Attaway DH, Mitsui A (1990) Japanese marine biotechnology: new opportunities for industrial microbiology. SIM News 40:45–51Google Scholar

Copyright information

© Springer-Verlag New York Inc 1992

Authors and Affiliations

  • H. L. Griffin
    • 1
  • R. V. Greene
    • 1
  • M. A. Cotta
    • 1
  1. 1.Biopolymer Research and Fermentation Biochemistry Research, National Center for Agricultural Utilization Research, U.S. Department of AgricultureAgricultural Research ServicePeoriaUSA

Personalised recommendations