Characterization of CpdC, a large-ring lactone-hydrolyzing enzyme from Pseudomonas sp. strain HI-70, and its use as a fusion tag facilitating overproduction of proteins in Escherichia coli

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DOIResolve DOI: http://doi.org/10.1128/AEM.02435-13
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TypeArticle
Journal titleApplied and Environmental Microbiology
ISSN0099-2240
Volume79
Issue22
Pages70917100; # of pages: 10
SubjectCarbon-carbon bond; Catalytic efficiencies; Degradation pathways; Expression in Escherichia coli; Global carbon cycle; Inclusion bodies; Pentadecalactone; Proof of concept; Bacteria; Dimers; Esters; Hydrolases; Proteins; Escherichia coli; bacterium; carbon cycle; coliform bacterium; enzyme activity; gene expression; homogeneity; hydrolysis; molecular analysis; plasmid; protein; solubilization
AbstractThere are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is aω-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone by Pseudomonas sp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (kcat) towardω-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (Tm) of CpdC was estimated to be 50.9±0.1°C. The half-life of CpdC at 35°C is several days. By virtue of its high level of expression in Escherichia coli, the intact CpdC-encoding gene and progressive 3=-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all in E. coli was demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form. © 2013, American Society for Microbiology.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); Aquatic and Crop Resource Development (ACRD-DCRA)
Peer reviewedYes
NPARC number21269828
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Record identifierd829a439-96a0-4919-b8e8-7257b3cff992
Record created2013-12-13
Record modified2016-05-09
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