Catalytic properties of variously immobilized mushroom tyrosinase: A kinetic study for future development of biomimetic amperometric biosensors

0524213 - ÚFCH JH 2021 RIV CH eng J - Journal Article
Sys, M. - Obluková, Michaela - Kolivoška, Viliam - Sokolová, Romana - Korecká, L. - Mikysek, T.
Catalytic properties of variously immobilized mushroom tyrosinase: A kinetic study for future development of biomimetic amperometric biosensors.
Journal of Electroanalytical Chemistry. Roč. 864, MAY 2020 (2020), č. článku 114066. ISSN 1572-6657. E-ISSN 1873-2569
R&D Projects: GA ČR(CZ) GA19-03160S
Institutional support: RVO:61388955
Keywords : carbon-paste electrodes * catecholase activity * complex * inactivation * cysteamine * catechin * chloride * Amperometry * Neurotransmitters * Enzyme immobilization * Enzyme kinetics * Mushroom tyrosinase * UV-Vis spectrophotometry
OECD category: Physical chemistry
Impact factor: 4.464, year: 2020
Method of publishing: Limited access

Mushroom tyrosinase was immobilized by direct embedding into electrode material (modified carbon paste electrode), incorporation of cross-linked enzyme aggregates into a polymer membrane (glassy carbon electrode covered by thin layer of Nafion (R)), and covalent attachment using self-assembled monolayers (gold electrode with the chemically bound enzyme). Both, standard UV-Vis spectrophotometry and amperometry in a batch configuration are presented as complementary methods to study the tyrosinase enzyme kinetics, whose catecholase activity results in electroactive products (ortho-quinones). Due to higher sensitivity of amperometric detection, evident advantage in the enzyme consumption was obtained. Prepared amperometric tyrosinase biosensors were characterized using cyclic voltammetry and atomic force microscopy. The Michaelis constant values of immobilized and unbound tyrosinase (free enzyme solution) towards dopamine and catechol were compared. The apparent Michaelis constant values for immobilized tyrosinase are significantly lower than the declared value of 0.840 mmol L-1 dopamine for the unbound enzyme. The enzymetyrosinase arranged in self-assembledmonolayer serves as an efficient sensor due to lowapparent Michaelis constant of 0.061 mmol L-1 dopamine and high maximum reaction velocity of 0.458 mu A s(-1). This fact reflects the ideal arrangement of enzymemolecules causing high availability of the binding site. Tris-glycine sodiumdodecyl sulphate polyacrylamide gel electrophoresis and atomic force microscopy clarified that the protein of molecular weight 25 kDa is bound preferably on chemically modified gold electrode. A sensor prepared by the immobilization of tyrosinase on gold electrode results in higher catecholase activity towards dopamine than in case of CPE and GC electrodes, where enzyme is immobilized physically.
Permanent Link: http://hdl.handle.net/11104/0308593