title: Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor.
abstract: A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 °C) when compared to PAMO in solution (no activity detected after 1 h at 60 °C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier.
authors.affiliation: Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands. Electronic address: m.krzek@rug.nl.
authors.forename: Marzena
authors.initials: M
authors.lastname: Krzek
authors.affiliation: Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands. Electronic address: h.van.beek@fz-juelich.de.
authors.forename: Hugo L
authors.initials: HL
authors.lastname: van Beek
authors.affiliation: University of Groningen, Department of Pharmacy, Analytical Biochemistry, Antonius-Deusinglaan 1, 9713 AV Groningen, The Netherlands. Electronic address: h.p.permentier@rug.nl.
authors.forename: Hjalmar P
authors.initials: HP
authors.lastname: Permentier
authors.affiliation: University of Groningen, Department of Pharmacy, Analytical Biochemistry, Antonius-Deusinglaan 1, 9713 AV Groningen, The Netherlands. Electronic address: r.p.h.bischoff@rug.nl.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
authors.affiliation: Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands. Electronic address: m.w.fraaije@rug.nl.
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D039903:Actinobacteria / Q000201:enzymology*; D001051:Apoenzymes / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D003067:Coenzymes / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D006358:Hot Temperature; D008863:Microspheres; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D003067:Coenzymes; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D012685:Sepharose; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase
keywords: Apo enzyme; FAD; Flavoenzyme; Immobilization; Monooxygenase