Hostname: page-component-586b7cd67f-2brh9 Total loading time: 0 Render date: 2024-11-26T13:44:10.087Z Has data issue: false hasContentIssue false
  • English
  • Français

Inhibition by chelating agents of the formation of active extracellular proteinase by Pseudomonas fluorescens 32A

Published online by Cambridge University Press:  01 June 2009

Robin C. Mckeller  and
Hilaire Cholette
Robin C. Mckeller
Affiliation:
Food Research Institute, Researcg Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada
Hilaire Cholette
Affiliation:
Food Research Institute, Researcg Branch, Agriculture Canada, Ottawa, Ontario K1A 0C6, Canada
Get access Rights & Permissions [Opens in a new window]

Summary

The effect of chelating agents on extracellular proteinase production by Pseudomonas fluorescens 32A was examined. Increasing concentrations of orthophosphate slightly stimulated growth while inhibiting proteinase synthesis. Fifty percent inhibition was found at 35 and 28 mM-orthophosphate at 5 and 20 °C respectively. Extracellular protein concentration was reduced by 30% when cells were grown with 100 mM-orthophosphate. Polyacrylamide gel electrophoresis of the cell-free supernatants suggested that reduced enzyme synthesis had taken place as evidenced by the decrease in staining intensity of the protein band corresponding to the proteinase. Other phosphate compounds could replace orthophosphate as an inhibitor. Extent of inhibition was related to chain length; polyphosphates with 4–6 or 13–18 phosphorus atoms were the most effective inhibitors. EDTA (0·5 mM) completely inhibited proteinase synthesis. This inhibition could be partly reversed by Ca2+ and, to a lesser extent, Mn2+. Proteinase production at 5 °C in skim milk was completely inhibited by phosphate glass (P13–P18). Control experiments showed that loss of activity with chelators was not due to inhibition of preformed enzyme. The results suggest a possible role for polyphosphates in controlling proteinase production in stored milk.

Type
Original Articles
Information
Journal of Dairy Research , Volume 52 , Issue 1 , February 1985 , pp. 91 - 100
Copyright
Copyright © Proprietors of Journal of Dairy Research 1985

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Adams, D. M., Barach, J. T. & Speck, M. L. 1975 Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin. Journal of Dairy Science 58 828–834CrossRefGoogle ScholarPubMed
Amrute, S. B. & Corpe, W. A. 1978 Production of extracellular protease by Pseudomonas fluorescens W. In Developments in Industrial Microbiology Vol. 19, pp. 465–471. (Ed. Underkefler, L. A.) New York: Plenum PressGoogle Scholar
Barach, J. T., Adams, D. M. & Speck, M. L. 1976 Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultra high temperature. Applied and Environmental Microbiology 31 875–879CrossRefGoogle Scholar
Cousin, M. A. 1982 Presence and activity of psychrotrophic micro–organisms in milk and dairy products: a review. Journal of Food Protection 45 172–207CrossRefGoogle Scholar
Davis, B. J. 1964 Disc electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences 121 404–427CrossRefGoogle ScholarPubMed
Driessen, F. M. 1981 Relationship between growth of Gram–negative rods in milk and their production of proteinase. Netherlands Milk and Dairy Journal 35 344–348Google Scholar
Harwalkar, V. R. & Vreeman, H. J. 1978 Effect of added phosphates and storage on changes in ultra-high-temperature short-time sterilized concentrated skim-milk. I. Viscosity, gelation, alcohol stability, chemical and electrophoretic analysis of proteins. Netherlands Milk and Dairy Journal 32 94–111Google Scholar
Juan, S. M. & Cazzulo, J. J. 1976 The extracellular protease from Pseudomonas fluorescens. Experientia 32 1120–1122CrossRefGoogle ScholarPubMed
Juffs, H. S. 1976 Effects of temperature and nutrients on proteinase production by Pseudomonas fluorescens and Ps. aeruginosa in broth and milk. Journal of Applied Bacteriology 40 23–32CrossRefGoogle ScholarPubMed
Juffs, H. S., Hayward, A. C. & Doelle, H. W. 1968 Growth and proteinase production in Pseudomonas spp. cultivated under various conditions of temperature and nutrition. Journal of Dairy Research 35 385–393CrossRefGoogle Scholar
Mckellar, R. C. 1982 Factors influencing the production of extracellular proteinase by Pseudomonas fluorescens. Journal of Applied Bacteriology 53 305–316CrossRefGoogle ScholarPubMed
Mckellar, R. C. & Cholette, H. 1984 Synthesis of extracellular proteinase by Pseudomonas fluorescens under conditions of limiting carbon, nitrogen, and phosphate. Applied and Environmental Microbiology 47 1224–1227CrossRefGoogle ScholarPubMed
Mikel'saar, P. Ch., Vilu, R. O. & Lakht, T. I. 1982 Synthesis of extracellular proteases as a function of the growth phase in Pseudomonas fluorescens. Mikrobiologiya 51 212–215Google ScholarPubMed
Murray, S. K., Kwan, K. K. H., Skura, B. J. & Mckellar, R. C. 1983 Effect of nitrogen flushing on the production of proteinase by psychrotrophic bacteria in raw milk. Journal of Food Science 48 1166–1169CrossRefGoogle Scholar
Odagiri, S. & Nickerson, T. A. 1964 Complexing of calcium by hexametaphosphate, oxalate, citrate and EDTA in milk. I. Effects of complexing agents on turbidity and rennet coagulation. Journal of Dairy Science 47 1306–1309CrossRefGoogle Scholar
Patel, T. R., Bartlett, F. M. & Hamid, J. 1983 Extracellular heat-resistant proteases of psychrotrophic pseudomonads. Journal of Food Protection 46 90–94CrossRefGoogle ScholarPubMed
Peterson, G. L. 1977 A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry 83 346–356CrossRefGoogle Scholar
Richardson, B. C. & Te Whaiti, I. E. 1978 Partial characterization of heat–stable extracellular proteases of some psychrotrophic bacteria from raw milk. New Zealand Journal of Dairy Science and Technology 13 172–176Google Scholar
Rowe, M. T. & Gilmour, A. 1982 Growth, enzyme production and changes in oxygen tension occurring during batch cultivation of psychrotrophic Pseudomonas fluorescens strains. Milchwissenschaft 37 597–600Google Scholar
Samel, R., Weaver, R. W. V. & Gammack, D. B. 1971 Changes on storage in milk processed by ultrahigh-temperature sterilization. Journal of Dairy Research 38 323–332CrossRefGoogle Scholar
Torrie, J. P., Cholette, H., Froehlich, D. A. & Mckellar, R. C. 1983 Growth of an extracellular proteinase–deficient strain of Pseudomonas fluorescens on milk and milk proteins. Journal of Dairy Research 50 365–374CrossRefGoogle ScholarPubMed
Vilu, R. O., Mikel'saar, P. Ch. & Lakht, T. I. 1980 Localization of metabolites regulating the synthesis of extracellular proteases in Pseudomonas fluorescens. Mikrobiologiya 49 708–714Google ScholarPubMed
Wilson, H. K., Vetter, J. L., Sasago, K. & Herreid, E. O. 1963 Effects of phosphates added to concentrated milks before sterilization at ultra-high-temperatures. Journal of Dairy Science 46 1038–1043CrossRefGoogle Scholar
Zemel, M. B. & Bidari, M. T. 1983 Zinc, iron and copper availability as affected by orthophosphates, polyphosphates and calcium. Journal of Food Science 48 567–569, 573CrossRefGoogle Scholar