Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes

Lipase from &x4domonas ueruginosu is a M, 29 kDa protein with a single functional disultide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Liited proteolysis of lipase with Staphylococcus uureus protease V8 resulted in cleavage after amino acid residues Asp)' and GIu~. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the al/l hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Se?, Asp*" and Hi?', and contains a disuhide bond connecting residues Cys'S) and C~S*~~. Residues Asp3* and Gl@ are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disultide bond could take place. A striking prediction from the model was the lack of a lid-like a-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.

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“…Lipases from I! cepacia [30] and E! glumae [31] belonging to homology group I also contain two Cys residues.…”

Section: Discussionmentioning

confidence: 99%

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

et al. 1993

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“…4), including the heptapeptide consensus motif (IGHSHGG) thought to lie at the active site of this family of lipases (Wohlfarth et al, 1992) (data not shown). A hlyC (iviXII)-containing mutant strain of V. cholerae that we constructed, AC-V195 (see below), lost the ability to produce a clearing zone around colonies grown for 48 h on agar media containing emulsified tributyrin (Jorgensen et al, 1991) (Fig. 5), thus demonstrating that the htyC (iviXII) locus is required for this secreted lipase activity.…”

Section: In Vivo-induced Locus Encoding a Secreted Lipasementioning

confidence: 99%

Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

Camilli

Mekalanos

1995

Molecular Microbiology

259

240

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SummaryA complete understanding of host-parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre-selection of strains carrying tnpR operon fusions (encoding resolvase, a site-specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvasespecific reporter construct. Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.

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“…Pseudomonas glumae was also found to be efficient in a system of washing model (Iizumi et al, 1991, Jorgesen et al, 1991. Span 80 was chosen for the following studies as it showed the highest percentage of oil removal in the presence of lipase.…”

Section: Resultsmentioning

confidence: 99%

Lipase from thermoalkalophilic Pseudomonas species as an additive in potential laundry detergent formulations

Khoo¹,

Ibrahim²

2009

MJM

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Lipase isolated from a thermoalkalophilic Pseudomonas species was used as additive to improve the degree of olive oil removal from cotton fabric in the presence of surfactants. The lipase used in this study was found to be more effective with non ionic surfactants as compared to ionic surfactants. In terms of stability, there was no decrease in activity found in the presence of Tween 85, Span 80 and Span 20. Lipase from Pseudomonas species was most active in the presence of Tween 85, Span 80 and Span 20. The application of lipase from Pseudomonas species as an additive in the formulation containing Span 80 has improved oil removal by 36% using the washing system consisting 5 U/mL lipase, at 70 °C for 20 min and 0.8% of Span 80 as surfactant. Considering that lipase from Pseudomonas species is stable in high pH and temperatures in the presence of various surfactants, therefore it is suitable to be incorporated as additives in potential detergent formulations.

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Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes

Cited by 147 publications

References 38 publications

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa

Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection

Lipase from thermoalkalophilic Pseudomonas species as an additive in potential laundry detergent formulations

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