Synthesis of functional unspecific peroxidases using a non-fungal eukaryotic cell-free system

The invention relates to a method for preparing a soluble and functional peroxygenase in cell-free systems which comprises at least the following steps:a) providing a reaction medium comprising at least the following components:a nucleic acid template coding for a fungal unspecific peroxygenase (UPO) classified as EC 111.2.1, an RNApolymerase, a eukaryotic cell lysate derived from insect cells or mammalian cells and supplemented with glutathione and glutathione-disulfide during lysate preparation wherein the cell lysate further comprises non-fungal membrane vesicles, as well as heme or a heme derivative, in particular a heme complex selected from the group comprising or consisting of hematin and hemin;b) performing a cell-free reaction to synthesize the peroxygenase protein.The invention further relates to a method for screening and characterization of mutants or variants of unspecific peroxygenases classified as EC 1.11.2.1., which method comprises steps a)- and b) of the method above and additionally at least a step c) screening the functional peroxygenase obtained after step b) for a target property, such as substrate specificity or stereoselectivity.A still further aspect of the invention relates to a reaction medium for preparing a soluble and functional peroxygenase in acell-free system comprising at least the following components: a nucleic acid template coding for a fungal unspecific peroxygenase (UPO) classified as EC 111.2.1, an RNApolymerase, a eukaryotic cell lysate derived from insect cells or mammalian cells and supplemented with glutathione and glutathione-disulfide during lysate preparation wherein the cell lysate further comprises non-fungal membrane vesicles and heme or a heme derivative, in particular a heme complex selected from the group comprising or consisting of hematin and hemin.