Reactor design for the hydroxylation of n-octane using a cyp153a6-based biocatalytic system expressed in Escherichia coli

Master Thesis

2013

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University of Cape Town

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Large stockpiles of linear hydrocarbons have arisen as by-products from the global expansion of gas-to-liquid refining processes. Furthermore, these linear alkanes feature one of the strongest chemical bonds in nature and typically are of a low value due to their inertness. In an effort to valorise this resource, catalytic routes are being sought in order to improve their value by introducing functional groups into the inert carbon backbone of such linear alkanes. Biocatalytic approaches have thus far provided the most feasible route for industrial applications of this chemistry as they feature a uniquely high selectivity for a vast range of products and operate under mild processing conditions. This study focuses on the biological hydroxylation of n-octane to 1-octanol using a previously developed cytochrome P450 monooxygenase, CYP153A6, enzymatic system. The biocatalyst was expressed in Escherichia coli BL21(DE3) by using a pET28b-PFR1500 plasmid encoding the complete operon from Mycobacterium sp. HXN-1500 which included the ferrodoxin reductase (FdR) and ferrodoxin (Fdx) redox partner proteins. CYP153A6 offers several benefits over other biocatalysts such as a notable !95 regioselectivity for terminal carbon hydroxylation and lack of product degradation through overoxidation and by-product formation. Studies to date focussing on understanding interactions between physiological, molecular and bioprocess conditions have yielded maximum specific biocatalyst activities and biocatalyst concentrations in the range of 4.0-5.5 μmoloctanol•gDCW−1•min−1 and 0.18 μmolP450•gDCW−1 respectively. Thus far, this particular P450-based biocatalytic system for n-octane hydroxylation has only been applied in small-scale vials. The objective of this work was to scale-up the experimental apparatus of this system from 1 ml (working volume) vials to bioreactors with a working volume in excess of 1 l because reactors afford improved process stability, control and easier analyses than small-scale experimental setups. The scope of this study focussed on aspects of process configuration and the scale-up for this process. More specifically, optimal timings with respect to induction of gene expression and alkane addition were ascertained with a focus on process stability. Molecular changes to this system were not considered.
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