Hostname: page-component-586b7cd67f-gb8f7 Total loading time: 0 Render date: 2024-11-23T07:11:56.520Z Has data issue: false hasContentIssue false
  • English
  • Français

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Published online by Cambridge University Press:  01 June 2009

David J. Fairbairn  and
Barry A. Law
David J. Fairbairn
Affiliation:
AFRC Institute of Food Research, Reading Laboratory (University of Reading), Shinfield, Reading RG2 9 AT, UK
Barry A. Law
Affiliation:
AFRC Institute of Food Research, Reading Laboratory (University of Reading), Shinfield, Reading RG2 9 AT, UK
Get access Rights & Permissions [Opens in a new window]

Summaey

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.

Type
Original Articles
Information
Journal of Dairy Research , Volume 53 , Issue 3 , August 1986 , pp. 457 - 466
Copyright
Copyright © Proprietors of Journal of Dairy Research 1986

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

Alichanidis, E. & Andrews, A. T. 1977 Some properties of the extracellular protease produced by the psychrotrophic bacterium Pseudomonas fluorescens strain AR-11. Biochimica et Biophysica Acta 485 424–433Google Scholar
Anderson, M. & Andrews, A. T. 1977 Progressive changes in individual milk protein concentrations associated with high somatic cell counts. Journal of Dairy Research 44 223–235Google Scholar
Barach, J. T. & Adams, D. M. 1977 Thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic Pseudomonas. Biochimica et Biophysica Acta 485 417–423CrossRefGoogle 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 ultrahigh temperature. Applied and Environmental Microbiology 31 875–879Google Scholar
Boethling, R. S. 1975 Purification and properties of a serine protease from Pseudomonas maltophilia. Journal of Bacteriology 121 933–941CrossRefGoogle Scholar
Bradford, M. M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248–254CrossRefGoogle ScholarPubMed
Carrick, L. & Berk, R. S. 1975 Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa. Biochimica et Biophysica Acta 391 422–434Google Scholar
Cliffe, A. J. & Law, B. A. 1982 A new method for the detection of microbial proteolytic enzymes in milk. Journal of Dairy Research 49 209–219CrossRefGoogle Scholar
Cliffe, A. J. & Law, B. A. 1985 Discontinuous polyacrylamide gel electrophoresis of cell wall proteinase from variants of Steptococcus lactis. Journal of Applied Bacteriology 58 245–250Google Scholar
Cousin, M. A. 1982 Presence and activity of psychrotrophic microorganisms in milk and dairy products: a review. Journal of Food Protection 45 172–207Google Scholar
Fairbairn, D. J. 1984 Production and properties of the extracellular proteinase from Pseudomonas fluorescent. Ph.D. Thesis, University of ReadingGoogle Scholar
Fairbairn, D. J. & Law, B. A. 1986 Proteinases of psychrotrophic bacteria: their production, properties, effects and control. Journal of Dairy Research 53 139–177Google Scholar
Jensen, S. E., Phillippe, L., Teng Tseng, J., Stemke, G. W. & Campbell, J. 1980 Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa. Canadian Journal of Microbiology 26 77–86Google Scholar
Law, B. A. 1980 Transport and utilization of proteins by bacteria. In Microorganisms and nitrogen sources, pp. 381–409, 783–786 (Ed. Payne, J. W.). New York: WileyGoogle Scholar
Law, B. A., Andrews, A. T. & Sharpe, M. E. 1977 Gelation of ultra-high-temperature-sterilized milk by proteases from a strain of Pseudomonas fluorescent isolated from raw milk. Journal of Dairy Research 44 145–148CrossRefGoogle Scholar
Law, B. A., Sharpe, M. E. & Chapman, H. R. 1976 The effect of lipolytic Gram-negative psychrotrophs in stored milk on the development of rancidity in Cheddar cheese. Journal of Dairy Research 43 459–468Google Scholar
Makino, K., Ogaki, J., Nichthara, T., Ichikawa, T. & Kondo, M. 1983 Studies on protease from marine bacteria. 2. Properties of extracellular protease from marine Pseudomonas sp. 145–2. Microbios 36 7–20Google Scholar
Mayerhofer, H. J., Marshall, R. T., White, C. H. & Lu, M. 1973 Characterization of a heat-stable protease of Pseudomonas fluorescens P26. Applied Microbiology 25 44–48Google Scholar
Morihara, K.,Tsuzuki, H.,Oka, T., Inoue, H. & Ebata, M. 1965 Pseudomonas aeruginosa elastase. Isolation, crystallization and preliminary characterization. Journal of Biological Chemistry 240 3295–3304Google Scholar
Noreau, J. & Drapeau, G. R. 1979 Isolation and properties of the protease from the wild-type and mutant strains of Pseudomonas fragi. Journal of Bacteriology 140 911–916Google Scholar
Ohta, Y.,Ooura, Y. & Wada, A. 1966 Thermostable protease from thermophilic, bacteria. I. Thermostability, physicochemical properties and amino acid composition. Journal of Biological Chemistry 241 5919–5929CrossRefGoogle Scholar
Patel, T. R., Jackman, D. M. & Bartlett, F. M. 1983 a Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization. Applied and Environmental Microbiology 46 333–337CrossRefGoogle ScholarPubMed
Patel, T. R., Bartlett, F. M. & Hamid, J. 1983 b Extracellular heat-resistant proteases of psychrotrophic pseudomonads. Journal of Food Protection 46 90–94, 97Google Scholar
Porzio, M. A. & Pearson, A. M. 1975 Isolation of an extracellular neutral proteinase from Pseudomonas fragi. Biochimica et Biophysica Acta 384 235–241Google Scholar
Richardson, B. C. 1981 The purification and characterization of a heat-stable protease from Pseudomonas fluorescens B52. New Zealand Journal of Dairy Science and Technology 16 195–207Google Scholar
Stepaniak, L. & Fox, P. F. 1983 Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36. Journal of Dairy Research 50 171–184Google Scholar
Stepaniak, L. & Fox, P. F. 1985 Isolation and characterization of heat-stable proteinases from Pseudomonas isolate AFT 21. Journal of Dairy Research 52 77–89Google Scholar
Stepaniak, L., Fox, P. F. & Daly, C. 1982 Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36. Biochimica et Biophysica Acta 717 376–383CrossRefGoogle ScholarPubMed
Voordouw, G. & Roche, R. S. 1974 The cooperative binding of two calcium ions to the double site of apothermolysin. Biochemistry 13 5017–5021Google Scholar
Wretlind, B. & Wadstrom, T. 1977 Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa. Journal of General Microbiology 103 319–327CrossRefGoogle ScholarPubMed