Purification of homogeneous forms of fungal peroxygenase.
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 until now been purified by several steps of fast protein liquid chromatography (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 pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were 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.
Ullrich
René
R
Unit of Environmental Biotechnology, International Graduate School of Zittau, Zittau, Germany. ullrich@ihi-zittau.de
Liers
Christiane
C
Schimpke
Sylvia
S
Hofrichter
Martin
M
D055362:Agrocybe / Q000737:chemistry / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification*; D009281:Naphthalenes / Q000378:metabolism; D020033:Protein Isoforms; D018892:Proton-Motive Force; D014050:Toluene / Q000378:metabolism
D005656:Fungal Proteins; D009281:Naphthalenes; D020033:Protein Isoforms; C031721:naphthalene; D014050:Toluene; D006899:Mixed Function Oxygenases; C022333:peroxygenase