title: Putrescine biosensor based on putrescine oxidase from Kocuria rosea.
abstract: The novel putrescine oxidase based amperometric biosensor selectively measures putrescine, which can be considered as an indicator of microbial spoilage. Putrescine oxidase (PUOX, EC 1.4.3.10) was isolated from Kocuria rosea (Micrococcus rubens) by an improved and simplified purification process. Cells were grown on brain heart infusion medium supplemented with putrescine. Cell-free extract was prepared in Tris buffer (pH 8.0) by Bead-beater. A newly elaborated step based on three-phase partitioning (TPP) was applied in the purification protocol of PUOX. The purified enzyme was immobilized on the surface of a spectroscopic graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinking agent. This modified working electrode was used in wall-jet type amperometric cell together with the Ag/AgCl (0.1M KCl) reference electrode and a platinum wire as auxiliary electrode in flow injection analysis system (FIA). Hydrogel composition, pH and potential dependence were studied. Optimal working conditions were 0.45 mLmin(-1) flow rate of phosphate buffer (66 mM, pH 8.0) and +50 mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25 mM putrescine, while the detection limit was 5 μM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.
authors.affiliation: EGERFOOD Regional Knowledge Centre, Eszterházy Károly College, Eszterházy tér 1, H-3300 Eger, Hungary. bokab@ektf.hu
authors.forename: Beáta
authors.initials: B
authors.lastname: Bóka
authors.forename: Nóra
authors.initials: N
authors.lastname: Adányi
authors.forename: Jenő
authors.initials: J
authors.lastname: Szamos
authors.forename: Diána
authors.initials: D
authors.lastname: Virág
authors.forename: Attila
authors.initials: A
authors.lastname: Kiss
mesh_terms: D001515:Beer / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D001709:Biotechnology / Q000379:methods*; D002244:Carbon; D003470:Culture Media; D004566:Electrodes; D008837:Micrococcus / Q000145:classification / Q000201:enzymology* / Q000254:growth & development; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification* / Q000378:metabolism; D011700:Putrescine / Q000032:analysis / Q000378:metabolism*; D011786:Quality Control
chemical_list: D003470:Culture Media; D002244:Carbon; C022488:putrescine oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011700:Putrescine