Allosteric Enzyme-Based Biosensors-Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties.

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme-substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

A novel approach for the selective analysis of l-lysine in untreated human serum by a co-crosslinked l-lysine-α-oxidase/overoxidized polypyrrole bilayer based amperometric biosensor.

An amperometric biosensor based on an l-lysine-α-oxidase (LO) layer immobilized by co-crosslinking onto the surface of an overoxidized polypyrrole modified Pt electrode (Pt/oPPy) and able to analyse l-lysine (Lys) in untreated human serum is described. The sensing electrode has been characterised and a proper enzyme kinetics optimisation permits to use a low specific enzyme as LO from Trichoderma viride for the selective biorecognition of Lys in the presence of other interferent amino acids; a kinetics study of LO evidenced also the allosteric behaviour of this enzyme, a kinetic feature which was never reported before for this enzyme. The biosensor showed a sensitivity of 0.11 µA/mM mm2, linear responses up to 4 mM and a limit of detection of 2 µM; the within-a-day coefficients of variation for replicate (n = 5) were 0.92% and 1.35% at 4 mM and 0.2 mM Lys levels, respectively. The permselective behaviour of Pt/oPPy modified electrode assured an interference- and fouling-free determination of Lys even in untreated serum samples. The determination of Lys in human serum from healthy donors gave Lys levels in good agreement with the expected values so that the use of the proposed biosensor appears promising in the relevant clinical fields.