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

Abstract:

: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.

authors

Bordeaux M,de Girval D,Rullaud R,Subileau M,Dubreucq E,Drone J

doi

10.1007/s00253-014-5671-1

subject

Has Abstract

pub_date

2014-01-01 00:00:00

pages

6275-83

issue

14

eissn

0175-7598

issn

1432-0614

journal_volume

98

pub_type

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