Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Catalysts

MarthaSophia Smit¹  Svenja Kochius¹  DiederikJohannes Opperman¹  AnaCristina Ebrecht¹  Jacqueline van Marwijk¹ 

[1] Department of Biotechnology, University of the Free State, Bloemfontein 9300, South Africa;

关键词: cytochrome P450 monooxygenase;    biocatalysis;    CYP153A6;    alkane hydroxylation;    cell-free extract biotransformations;    1-octanol;    cofactor regeneration;   

DOI  :  10.3390/catal8110531

来源: DOAJ

【 摘 要 】

Some of the most promising results for bacterial alkane hydroxylation to alcohols have been obtained with the cytochrome P450 monooxygenase CYP153A6. CYP153A6 belongs to the class I CYPs and is generally expressed from an operon that also encodes the ferredoxin (Fdx) and ferredoxin reductase (FdR) which transfer electrons to CYP153A6. In this study, purified enzymes (CYP, Fdx, FdR and dehydrogenases for cofactor regeneration) were used to deconstruct the CYP153A6 system into its separate components, to investigate the factors limiting octane hydroxylation in vitro. Proteins in the cytoplasm (cell-free extract) were found to better enhance and stabilize hydroxylase activity compared to bovine serum albumin (BSA) and catalase. Optimization of the CYP:Fdx:FdR ratio also significantly improved both turnover frequencies (TFs) and total turnover numbers (TTNs) with the ratio of 1:1:60 giving the highest values of 3872 h⁻¹ and 45,828 mol_octanol mol_CYP⁻¹, respectively. Choice and concentration of dehydrogenase for cofactor regeneration also significantly influenced the reaction. Glucose dehydrogenase concentrations had to be as low as possible to avoid fast acidification of the reaction medium, which in the extreme caused precipitation of the CYP and other proteins. Cofactor regeneration based on glycerol failed, likely due to accumulation of dihydroxyacetone. Scaling the reactions up from 1 mL in vials to 60 mL in shake flasks and 120 mL in bioreactors showed that mixing and shear forces will be important obstacles to overcome in preparative scale reactions.

【 授权许可】

Unknown