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

Abstract

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 Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine

“…In flow injection approaches using an amperometric biosensor as in the present case, flow rate is well-known to rule the apparent enzyme kinetics and the measured currents [60]. In this respect, Figure S3 reports the flow rate dependences for Ch and PCh at the dual amperometric biosensor.…”

Discriminative detection of glucose and urea with a composite polymer nanofiber based matrix

“…[85] It has been employed for evaluating inhibition effects [86] or cooperativity dependent on external factors such as pH. [87] Since we immobilized two enzyme molecules together in close proximity, a pseudo-allosteric behaviour may be present, thus, we thought it relevant to analyze the Hill plot. For both mono-and bienzymatic platforms, the parallel linear plots exhibited slopes equivalent to n = Hill coefficient, of 1.02 (R 2 = 0.992) for Urease and 1.01 (R 2 = 0.995) for Urease-GOD (Figure 4b), thus revealing no effects in terms of competitive or cooperant effect.…”

Stimuli-Responsive Regulation of Biocatalysis through Metallic Nanoparticle Interaction

“…Redox biomolecules, such as certain enzymes, were first immobilized on electrode surfaces almost six decades ago, aiming mainly for biosensor applications . Despite the growth of the biosensor field as a hot topic, − bioelectrochemistry currently presents a more diverse range of applications, such as biofuel cells, − biosupercapacitors, − and bioreactors. − The development of nanotechnology and, hence, its application in surface-modified electrodes has been demonstrated to be a versatile tool. In particular, a conjugated system of metallic nanoparticles and enzymes is able to provide increased surface area, which bears the immobilization of higher loads of enzyme, and thus, higher current densities can be reached in electrochemical assays .…”

“…The specific control of biocatalyst properties as well as self-controlled regulation have been a great desire for scientists. Understanding how to mimic biocatalytic processes in nature may be helpful to combat diseases or to improve biocatalytic events in the areas of biomedicine, ,, biosensors, − industry, and energy, − among many others. Over the past few years, major progress has been made to develop remote regulation of enzymes, and the use of metallic NPs certainly offers a great opportunity in this direction.…”