Putrescine biosensor based on putrescine oxidase from Kocuria rosea

Beáta Bóka, N. Adányi, Jeno Szamos, Diána Virág, Attila Kiss

Research output: Article

22 Citations (Scopus)

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 0.45mLmin-1 flow rate of phosphate buffer (66mM, pH 8.0) and +50mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25mM putrescine, while the detection limit was 5μM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.

Original languageEnglish
Pages (from-to)258-262
Number of pages5
JournalEnzyme and Microbial Technology
Volume51
Issue number5
DOIs
Publication statusPublished - okt. 10 2012

Fingerprint

putrescine oxidase
Putrescine
Biosensing Techniques
Biosensors
Electrodes
Hydrogels
Purification
Hydrogel
Spoilage
Graphite electrodes
Flow Injection Analysis
Micrococcus
Crosslinking
Immobilized Enzymes
Tromethamine
Polyethylene glycols
Graphite
Platinum
Ethers
Brain

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Putrescine biosensor based on putrescine oxidase from Kocuria rosea. / Bóka, Beáta; Adányi, N.; Szamos, Jeno; Virág, Diána; Kiss, Attila.

In: Enzyme and Microbial Technology, Vol. 51, No. 5, 10.10.2012, p. 258-262.

Research output: Article

Bóka, Beáta ; Adányi, N. ; Szamos, Jeno ; Virág, Diána ; Kiss, Attila. / Putrescine biosensor based on putrescine oxidase from Kocuria rosea. In: Enzyme and Microbial Technology. 2012 ; Vol. 51, No. 5. pp. 258-262.
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