Engineering unspecific peroxygenases by structure-guided in vivo recombination of homologous protein blocks

ABSTRACT

Fungal unspecific peroxygenases (UPOs) are highly versatile enzymes for C-H oxyfunctionalization reactions. Over the years, they have been subjected to directed evolution campaigns in order to improve heterologous functional expression, activity, stability and selectivity. While the number of UPO genes available for protein engineering is steadily increasing, their use in enzyme chimeragenesis experiments has been little explored. In this chapter we describe how to construct functionally diverse UPO chimeras from different orthologs by applying the SCHEMA-RASPP computational algorithm in combination with in vivo DNA shuffling.