High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and mode...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2014, Vol.98 (14), p.6275-6283
Main Authors: Bordeaux, Mélanie, de Girval, Diane, Rullaud, Robin, Subileau, Maeva, Dubreucq, Eric, Drone, Jullien
Format: Article
Language:English
Subjects:
Alcanivoraceae - enzymology
Alcanivoraceae - genetics
Alcanivorax
Biomedical and Life Sciences
Biotechnological Products and Process Engineering
Biotechnology
Chemical properties
Cytochrome P-450 CYP4A - genetics
Cytochrome P-450 CYP4A - isolation & purification
Cytochrome P-450 CYP4A - metabolism
Enzymes
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli - growth & development
Genetic aspects
Hydrolases
Life Sciences
Microbial Genetics and Genomics
Microbiology
Octanes - metabolism
Physiological aspects
Properties
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
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Summary:We have recently described the biocatalytic characterization of a self-sufficent biosynthetic alkane hydroxylase based on CYP153A13a from Alcanivorax borkumensis SK2 (thereafter A13-Red). Despite remarkable regio- and chemo-selectivity, A13-Red suffers of a difficult-to-reproduce expression and moderate operational stability. In this study, we focused our efforts on the production of A13-Red using high-cell-density cultivation (HCDC) of recombinant Escherichia coli . We achieved 455 mg (5,000 nmol) of functional enzyme per liter of culture. Tight control of cultivation parameters rendered the whole process highly reproducible compared with flask cultivations. We optimized the purification of the biocatalyst that can be performed in either two or three steps depending on the application needed to afford A13-Red up to 95 % homogeneous. We investigated different reaction conditions and found that the total turnover numbers of A13-Red during the in vitro hydroxylation of n -octane could reach up to 3,250 to produce 1-octanol (1.6 mM) over a period of 78 h.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-014-5671-1