Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli.
BACKGROUND
Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).


RESULTS
Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from approximately 500 up to approximately 25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies.


CONCLUSIONS
Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in E. coli or Streptomyces spp.), shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.
Volontè
Federica
F
Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria via JH Dunant 3, 21100 Varese, Italy.
Pollegioni
Loredano
L
Molla
Gianluca
G
Frattini
Luca
L
Marinelli
Flavia
F
Piubelli
Luciano
L
D000595:Amino Acid Sequence; D019149:Bioreactors; D001951:Brevibacterium / Q000201:enzymology*; D002789:Cholesterol Oxidase / Q000096:biosynthesis* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary; D004926:Escherichia coli / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D003470:Culture Media; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D002789:Cholesterol Oxidase
Nature. 1970 Aug 15;227(5259):680-5
5432063
Lett Appl Microbiol. 1993 Aug;17(2):61-4
7763933
FEBS J. 2009 Dec;276(23):6857-70
19843167
J Biol Chem. 2008 Sep 5;283(36):24738-47
18614534
FEBS J. 2009 Dec;276(23):6844-56
19843168
J Biol Chem. 2001 May 25;276(21):18024-30
11359791
Appl Microbiol Biotechnol. 2001 Oct;57(1-2):146-52
11693912
J Biol Chem. 2001 Aug 10;276(32):30435-41
11397813
Appl Environ Microbiol. 1986 Dec;52(6):1382-5
3466572
Biochem J. 2006 Nov 15;400(1):13-22
16856877
Protein Sci. 2005 Dec;14(12):3064-76
16260759
J Biol Chem. 2005 Jun 17;280(24):22572-81
15817448
FEBS J. 2009 Dec;276(23):6826-43
19843169
J Steroid Biochem Mol Biol. 2000 Apr;72(5):169-95
10822008
J Biosci Bioeng. 2001;92(5):459-65
16233128
Biotechnol Lett. 2008 Jan;30(1):165-72
17849088
Biosci Biotechnol Biochem. 1992 Nov;56(11):1786-91
1369073
Biotechnol Lett. 2007 May;29(5):761-6
17237971
Antioxid Redox Signal. 2001 Oct;3(5):789-806
11761328
FEMS Microbiol Lett. 2002 Oct 8;215(2):243-8
12399041
J Biol Chem. 2004 Jan 2;279(1):95-102
14555654
Eur J Biochem. 1999 Aug;264(1):140-51
10447682
Protein Expr Purif. 1998 Apr;12(3):347-52
9535702
Appl Microbiol Biotechnol. 2009 Mar;82(3):479-90
19015844