Purification and properties of extracellular lipase from EF2 Free

Abstract

Summary: Extracellular lipase was purified from a Tween 80-limited continuous culture of EF2 by ultrafiltration of the culture supernatant followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit ( 29000, pI 4·9), which was capable of a variable degree of aggregation, and which exhibited both lipase activity, measured with the insoluble substrate olive oil (predominantly triolein), and esterase activity, measured with the soluble substrates -nitrophenyl acetate and Tween 80. Lipase activity was approximately eight times higher than either type of esterase activity ( , approximately 3000 sfor the hydrolysis of olive oil). The enzyme showed a marked regiospecificity for the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures (exhibiting a biphasic loss of activity with an initial of 17·5 min at 60 °C) and was very stable to freezing and thawing. Lipase activity was only weakly inhibited by the serine-active reagent 3,4-dichloroisocoumarin, and was not inhibited by the chelating agent EDTA (1 mm). The N-terminal amino acid sequence of the EF2 lipase showed a marked similarity to those of several other bacterial lipases.

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1991-09-01
2024-03-29
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References

  1. Andreoli P. M., Cox M. M. J., Farin F., Wohlfarth S. 1989; Molecular cloning and expression of genes encoding lipolytic enzymes. European Patent 0334462
    [Google Scholar]
  2. Aoyama S., Yoshida N., Inouye S. 1988; Cloning, sequencing and expression of the lipase gene from Pseudomonas fragi IFO-12049 in E. coli . FEBS Letters 242:36–40
    [Google Scholar]
  3. Bozoǧlu F., Swaisgood H. E., Adams D. M. 1984; Isolation and characterisation of an extracellular heat-stable lipase produced by Pseudomonas fluorescens MC 50. Journal of Agricultural and Food Chemistry 32:2–6
    [Google Scholar]
  4. Brockerhoff H., Jensen R. G. 1974 Lipolytic Enzymes New York: Academic Press;
    [Google Scholar]
  5. Dring R., Fox P. F. 1983; Purification and characterisation of a heat-stable lipase from Pseudomonas fluorescens AFT 29. Irish Journal of Food Science and Technology 7:157–171
    [Google Scholar]
  6. Fox P. F., Stepaniak L. 1983; Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36. Journal of Dairy Research 50:77–89
    [Google Scholar]
  7. Gilbert E. J., Drozd J. W., Jones C. W. 1991; Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2. Journal of General Microbiology 137:2215–2221
    [Google Scholar]
  8. Hames B. D. 1981; An introduction to polyacrylamide gel electro-phoresis. Gel Electrophoresis of Proteins1–91 Hames B. D., Rickwood D. Oxford : IRL Press;
    [Google Scholar]
  9. Harper J. W., Hemmi K., Powers J. C. 1985; Reaction of serine proteases with substituted isocoumarins : discovery of 3,4-dichloro-isocoumarin, a new general mechanism based serine protease inhibitor. Biochemistry 24:1831–1841
    [Google Scholar]
  10. Harwood J. 1989; The versatility of lipases for industrial uses. Trends in Biochemical Sciences 14:125–126
    [Google Scholar]
  11. Jäger K. E., Wohlfarth S., Winkler U. K. 1991; Extracellular lipase of Pseudomonas aeruginosa . Lipases: Structure, Mechanism and Genetic Engineering (GBF monograph no. 16)381–384 Alberghina L., Verger R., Schmid R. D. Weinheim: VCH;
    [Google Scholar]
  12. Jørgensen S., Skov K. W., Diderichsen B. 1991; Cloning, sequence and expression of a lipase gene from Pseudomonas cepacia : lipase production in heterologous hosts requires two Pseudomonas genes. Journal of Bacteriology 173:559–567
    [Google Scholar]
  13. Kordel M., Schmid R. D. 1991; Inhibition of the lipase from Pseudomonas sp. ATCC 21808 by diethyl p-nitrophenyl phosphate; hints for one buried active site for lipolytic and esterolytic activity. Lipases: Structure, Mechanism and Genetic Engineering (GBF monographs no. 16)385–388 Alberghina L., Verger R., Schmid R. D. Weinheim; VCH:
    [Google Scholar]
  14. Kugimiya W., Otanis Y., Hashimoto Y., Tagaki Y. 1986; Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi . Biochemical and Biophysical Research Communications 141:185–190
    [Google Scholar]
  15. Lee C. Y., Iandolo J. J. 1986; Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. Journal of Bacteriology 166:385–391
    [Google Scholar]
  16. Macrae A. R. 1983; Extracellular microbial lipases. Microbial Enzymes and Biotechnology225–250 Fogarty W. M. London: Applied Science Publishers;
    [Google Scholar]
  17. Macrae A. R., Hammond R. C. 1985; Present and future applications of lipases. Biotechnology and Genetic Engineering Reviews 3:193–217
    [Google Scholar]
  18. Nakanishi J., Kurono Y., Kolde Y., Beppu T. 1989; Recombinant DNA, bacterium of the genus Pseudomonas containing it, and process for preparing lipase using it. European Patent 0331376
    [Google Scholar]
  19. Nishioka T., Chihara-Shiomi M., ., Yoshikawa K., Inagaki M., Yamamoto Y., Hiratake J., Baba N., Oda J. 1991; Lipase from Pseudomonas sp. : reactions, cloning and amino acid sequence analysis. Lipases: Structure, Mechanism and Genetic Engineering (GBF monographs No. 16)253–262 Alberghina L., Verger R., Schmid R. D. Weinheim: VCH;
    [Google Scholar]
  20. Okumura S., Iwai M., Tsujisaka Y. 1976; Positional specificities of four kinds of microbial lipases. Agricultural and Biological Chemistry 40:655–660
    [Google Scholar]
  21. van Oort M. G., Deever A. M. T. J., Dijkman R., Tjeenk M. L., Verheij H. M., de Haas G. H., Wenzig E., Gotz F. 1989; Purification and substrate specificity of Staphylococcus hyicus lipase. Biochemistry 28:9278–9285
    [Google Scholar]
  22. Silman N. J., Carver M. A., Jones C. W. 1989; Physiology of amidase production by Methylophilus methylotrophus: isolation of hyperactive strains using continuous culture. Journal of General Microbiology 135:3153–3164
    [Google Scholar]
  23. Silan N. J., Carver M. A., Jones C. W. 1991; Directed evolution of amidase in Methylophilus methylotrophus: purification and properties of amidases from wild-type and mutant strains. Journal of General Microbiology 137:169–178
    [Google Scholar]
  24. Stuer W., Jaeger K. E., Winkler U. K. 1986; Purification of extracellular lipase from Pseudomonas aeruginosa . Journal of Bacteriology 168:1070–1074
    [Google Scholar]
  25. Yamamoto K., Fujiwara N. 1988; Purification and some properties of a castor-oil hydrolysing lipase from Pseudomonas sp. Agricultural and Biological Chemistry 52:3015–3021
    [Google Scholar]
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