Iwona Kaluzna, Jan Brummund, Martin Schuermann
InnoSyn B.V., Geleen, The Netherlands

Abstract

Cytochrome P450 monooxygenases can catalyze highly regio- and stereo-selective oxidations of many organic compounds. Nevertheless, the scale-up of such biooxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges the demand for these recombinant biocatalysts is increasing, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4′- and 5-hydroxy metabolites. Here we report selective hydroxylation of diclofenac to 4’-hydroxy- and 5-hydroxy-diclofenac on multi hundred-gram scale.

INTRODUCTION

Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereo-specific oxidations of a broad substrate range of small organic molecules. However, the application of this enzyme class on large scale is still very challenging due to various aspects such as limited enzyme activities and stability, hydrophobicity of the substrates or the necessity of an efficient electron and oxygen supply to the biocatalyst and the biooxidation reactor. Despite these challenges the demand for these recombinant biocatalysts is increasing (1), particularly for the production of drug metabolites, due to their ability of oxygen introduction into non-activated C-H bonds. Here, we demonstrate the feasibility of cytochrome P450 technology to produce metabolites on gram scale. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4’- and 5-hydroxy metabolites (2).
Here we report the selective hydroxylation of diclofenac to 4’-hydroxy- and 5-hydroxy-diclofenac on multi hundred-gram scale using a recombinantly produced variant of the bacterial P450 CYP102A1 from