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. 1995 Mar;177(5):1254–1258. doi: 10.1128/jb.177.5.1254-1258.1995

Lipase modulator protein (LimL) of Pseudomonas sp. strain 109.

F Ihara 1, I Okamoto 1, K Akao 1, T Nihira 1, Y Yamada 1
PMCID: PMC176731  PMID: 7868599

Abstract

Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH2-terminal sequence and a lipase modulator gene (limL) lacking the NH2-terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH2-terminal amino acid sequence was determined to be NH2-Leu-Glu-Pro-Ser-Pro-Ala-Pro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.

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Selected References

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  1. Chihara-Siomi M., Yoshikawa K., Oshima-Hirayama N., Yamamoto K., Sogabe Y., Nakatani T., Nishioka T., Oda J. Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa. Arch Biochem Biophys. 1992 Aug 1;296(2):505–513. doi: 10.1016/0003-9861(92)90604-u. [DOI] [PubMed] [Google Scholar]
  2. Frenken L. G., Bos J. W., Visser C., Müller W., Tommassen J., Verrips C. T. An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase. Mol Microbiol. 1993 Aug;9(3):579–589. doi: 10.1111/j.1365-2958.1993.tb01718.x. [DOI] [PubMed] [Google Scholar]
  3. Frenken L. G., de Groot A., Tommassen J., Verrips C. T. Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae. Mol Microbiol. 1993 Aug;9(3):591–599. doi: 10.1111/j.1365-2958.1993.tb01719.x. [DOI] [PubMed] [Google Scholar]
  4. Gilbert E. J. Pseudomonas lipases: biochemical properties and molecular cloning. Enzyme Microb Technol. 1993 Aug;15(8):634–645. doi: 10.1016/0141-0229(93)90062-7. [DOI] [PubMed] [Google Scholar]
  5. Hobson A. H., Buckley C. M., Aamand J. L., Jørgensen S. T., Diderichsen B., McConnell D. J. Activation of a bacterial lipase by its chaperone. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5682–5686. doi: 10.1073/pnas.90.12.5682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ihara F., Kageyama Y., Hirata M., Nihira T., Yamada Y. Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species. J Biol Chem. 1991 Sep 25;266(27):18135–18140. [PubMed] [Google Scholar]
  7. Iizumi T., Nakamura K., Shimada Y., Sugihara A., Tominaga Y., Fukase T. Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56. Agric Biol Chem. 1991 Sep;55(9):2349–2357. [PubMed] [Google Scholar]
  8. Jørgensen S., Skov K. W., Diderichsen B. Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes. J Bacteriol. 1991 Jan;173(2):559–567. doi: 10.1128/jb.173.2.559-567.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mizobata T., Akiyama Y., Ito K., Yumoto N., Kawata Y. Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli. Refolding is enhanced in the presence of ADP. J Biol Chem. 1992 Sep 5;267(25):17773–17779. [PubMed] [Google Scholar]
  10. Oshima-Hirayama N., Yoshikawa K., Nishioka T., Oda J. Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene. Eur J Biochem. 1993 Jul 15;215(2):239–246. doi: 10.1111/j.1432-1033.1993.tb18028.x. [DOI] [PubMed] [Google Scholar]
  11. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  13. Wohlfarth S., Hoesche C., Strunk C., Winkler U. K. Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1. J Gen Microbiol. 1992 Jul;138(7):1325–1335. doi: 10.1099/00221287-138-7-1325. [DOI] [PubMed] [Google Scholar]
  14. Zaks A., Klibanov A. M. Enzymatic catalysis in organic media at 100 degrees C. Science. 1984 Jun 15;224(4654):1249–1251. doi: 10.1126/science.6729453. [DOI] [PubMed] [Google Scholar]
  15. Zaks A., Klibanov A. M. Enzyme-catalyzed processes in organic solvents. Proc Natl Acad Sci U S A. 1985 May;82(10):3192–3196. doi: 10.1073/pnas.82.10.3192. [DOI] [PMC free article] [PubMed] [Google Scholar]

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