Thermostable Proteins as Probe for the Design of Advanced Fluorescence Biosensors

Abstract

In this review we explore the advantages deriving from the use of either enzymes or sugar binding proteins isolated from thermophilic organisms to develop stable fluorescence biosensors. We report on a novel approach to address the consumption of the analyte by enzyme-based biosensors, namely the utilization of apo-enzymes as non-active forms of proteins which are still able to bind the ligand but cannot transform it into product. We also report recent studies in which the fluorescence labeling of a naturally thermostable binding protein allows a quantitative determination of glucose. Abbreviations IAANS 2-(4¢-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) ANS 1-(anilino)-naphtalene-8-sulfonate FRET fluorescence resonance energy transfer LED light emitting diode GD glucose dehydrogenase BSGK glucokinase from Bacillus stearothermophilus Ph-SBP sugar-binding protein from Pyrococcus horikoshii M. de Champdoré AE M. Staiano AE V. Aurilia AE

Confocal imaging of protein distributions in porous silicon optical structures

“…The labelled protein used to image the PSi inner distribution is the D-trehalose/D-maltose-binding protein (TMBP) is one component of the trehalose (Tre) and maltose (Mal) uptake system which, in the hyperthermophilic archaeon T. litoralis, is mediated by a protein-dependent ATP-binding cassette (ABC) system transporter [15]. TMBP from T. litoralis is a monomeric 48 kDa two-domain macromolecule containing 12 tryptophan residues [16]. Details of purification, preparation, and labelling of TMBP can be found in the literature [17].…”

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

“…They display exceptional stability and optimum activity at high temperature, making them favored industrial enzymes. Reports of thermophilic enzymes being used as diagnostic biosensors also exist (7); however, their applicability in therapeutic purposes remains far from reach. This is because enzymes for therapeutic purposes should work ideally at physiological conditions, while thermophilic enzymes act optimally at high temperatures.…”

Detection and Monitoring of Microparticles Under Skin by Optical Coherence Tomography as an Approach to Continuous Glucose Sensing Using Implanted Retroreflectors

“…Methods including kinetic enzymatic assays using glucose oxidation [19], [20], chemical binding of glucose to synthetic boronic acid [21], [22], affinity binding to Concanavalin A (Con A) [23]–[25], and affinity binding to glucose binding proteins [26], [27] have been studied and combined with fluorescence, scattering and fiber optic techniques for blood glucose sensing. Glucose recognition utilizing affinity binding to Con A offers certain advantages, including full reversibility, feasibility of calibration, weak pH-dependence, and no effect of varying oxygen partial pressure [5].…”