title: Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.
abstract: Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Zernia
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Ott
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bellmann-Sickert
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.|Centre for Biotechnology and Biomedicine, Leipzig University , Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Frank
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.|Centre for Biotechnology and Biomedicine, Leipzig University , Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Klenner
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.|Centre for Biotechnology and Biomedicine, Leipzig University , Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Heinz-Georg
authors.initials: HG
authors.lastname: Jahnke
authors.affiliation: Leibniz-Institute of Surface Modification (IOM) , Permoserstraße 15, 04318 Leipzig, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Prager
authors.affiliation: Leibniz-Institute of Surface Modification (IOM) , Permoserstraße 15, 04318 Leipzig, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Abel
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.|Centre for Biotechnology and Biomedicine, Leipzig University , Deutscher Platz 5, 04103 Leipzig, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Robitzki
authors.affiliation: Institute of Biochemistry, Leipzig University , Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System