Purification of homogeneous forms of fungal peroxygenase

René Ullrich (1) , Christiane Liers (1) , Sylvia Schimpke (2) , Martin Hofrichter (1)

1 Environmental Biotechnology
2 Biotechnology Center and Center for Regenerative Therapies

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has been purified, so far, by several steps of FPLC using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different isoelectric points. All AaP forms oxygenated toluene and naphthalene but catalytic differences were not observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.

Mots clés: Life Sciences

René Ullrich, Christiane Liers, Sylvia Schimpke, Martin Hofrichter. Purification of homogeneous forms of fungal peroxygenase. Biotechnology Journal, 2009, 4 (11), pp.1619. ⟨10.1002/biot.200900076⟩. ⟨hal-00498913⟩