Oxidative dehalogenation using the unspecific peroxygenase variant PaDa-I

Sammanfattning

Unspecific peroxygenases (UPOs) are a group of enzymes that catalyse peroixidase- and peroxygenase reactions. This enables them to carry out oxyfunctionalization, the valorization of inert molecules through oxygen insertion. They distinguish themselves from other enzymes capable of the same chemistry (e.g. cytochrome P450s) through their ability to run cofactor free catalysis. The enzyme variant PaDa-I, evolved for higher activity, secretability and stability, was screened for oxidative dehalogenation activity, a reaction not catalogued for any UPO. Five substrates were screened: benzyl bromide, benzyl chloride, 4-nitrobenzyl chloride, 1-(1-chloroethyl)-4-nitrobenzene and 2-chloroheptane. All aromatic substrates were successfully converted to corresponding dehalogenated, turning primary- and secondary halogen positions into aldehydes and ketones respectively. Upscaling the dehalogenation of chlorinated substrates demonstrated a procedure bottleneck, the chloride expelled from the substrate during oxidation becomes a new substrate for the UPO and becomes subsequently oxidized into hypochlorite (ClO-) which inactivates PaDa-I. Attempts to sequester chloride and limit hypochlorite formation using silver nitrate did not increase yields and instead lead to faster enzyme inactivation. This instead lead to increased insight about potential bottlenecks for future development of UPO catalysed oxidative dehalogenation. A pH and buffer dependency assay displayed no significant activity differences between buffers but strong pH dependent effects on the reaction rate with a maximum at pH 5.0.