title: 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.
abstract: There 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 a ω-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) toward ω-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.
authors.affiliation: Aquatic and Crop Resource Development, National Research Council Canada, Montreal, Quebec, Canada.
authors.forename: Yali
authors.initials: Y
authors.lastname: Xu
authors.forename: Stephan
authors.initials: S
authors.lastname: Grosse
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Iwaki
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D002479:Inclusion Bodies / Q000201:enzymology; D018942:Macrolides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D018942:Macrolides; C571591:omega-pentadecalactone; D006867:Hydrolases; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
references.citation: Appl Environ Microbiol. 1999 Nov;65(11):5158-62
references.pmid: 10543838
references.citation: Protein Eng. 1992 Apr;5(3):197-211
references.pmid: 1409539
references.citation: Gene. 1985;33(1):103-19
references.pmid: 2985470
references.citation: Appl Environ Microbiol. 2006 Apr;72(4):2707-20
references.pmid: 16597975
references.citation: Biochem Biophys Res Commun. 2009 Feb 6;379(2):553-6
references.pmid: 19116143
references.citation: Mol Biol Evol. 2011 Oct;28(10):2731-9
references.pmid: 21546353
references.citation: Appl Microbiol Biotechnol. 1999 Jul;52(1):85-90
references.pmid: 10461374
references.citation: Chem Rev. 2004 Sep;104(9):4105-24
references.pmid: 15352787
references.citation: Appl Microbiol Biotechnol. 2012 Jan;93(1):229-40
references.pmid: 21713515
references.citation: J Bacteriol. 2001 Nov;183(21):6478-86
references.pmid: 11591693
references.citation: Chembiochem. 2010 Nov 2;11(16):2208-31
references.pmid: 20936617
references.citation: Biotechnol J. 2011 Oct;6(10):1230-9
references.pmid: 21751386
references.citation: Adv Appl Microbiol. 2009;66:77-95
references.pmid: 19203649
references.citation: Microb Biotechnol. 2010 Jan;3(1):65-73
references.pmid: 21255307
references.citation: Methods Enzymol. 2009;463:259-82
references.pmid: 19892177
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14098-103
references.pmid: 12393814
references.citation: Nat Biotechnol. 2006 Jan;24(1):100-4
references.pmid: 16369539
references.citation: Appl Microbiol Biotechnol. 2008 Nov;81(1):79-87
references.pmid: 18758768
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D423-9
references.pmid: 23193256
references.citation: Chem Rev. 2011 Jul 13;111(7):4165-222
references.pmid: 21542563
references.citation: J Biol Chem. 2008 Sep 19;283(38):25854-62
references.pmid: 18658138
references.citation: J Am Chem Soc. 2009 Jul 1;131(25):8848-54
references.pmid: 19385644
references.citation: Appl Microbiol Biotechnol. 2003 Jan;60(5):523-33
references.pmid: 12536251
references.citation: Nat Biotechnol. 2004 Nov;22(11):1399-408
references.pmid: 15529165
references.citation: Infect Immun. 1997 Jan;65(1):16-23
references.pmid: 8975887
references.citation: Appl Environ Microbiol. 2002 Nov;68(11):5671-84
references.pmid: 12406764
references.citation: Appl Microbiol Biotechnol. 2006 Sep;72(2):211-22
references.pmid: 16791589
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: Biochemistry. 1996 Jul 9;35(27):8805-14
references.pmid: 8688416
references.citation: J Am Chem Soc. 2009 Nov 4;131(43):15711-6
references.pmid: 19810726
references.citation: Microb Cell Fact. 2013 May 02;12:43
references.pmid: 23638724
references.citation: Microb Cell Fact. 2012 Jan 12;11:8
references.pmid: 22239833
references.citation: Trends Biotechnol. 2012 Feb;30(2):65-70
references.pmid: 22037492
references.citation: Appl Microbiol Biotechnol. 2002 May;58(6):781-9
references.pmid: 12021799
references.citation: J Bacteriol. 2000 Sep;182(17):4744-51
references.pmid: 10940013