Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States

Abstract

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase-the enzyme responsible for allantoin hydrolization in plants and lower organisms-for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Protein‐based metal bio‐cleaner for detoxification of wastewater

“…Hence, the presence of specific metals in a solution or biological system can be detected by the metal‐induced changes in the fluorescence of fluorescent protein‐based biosensors. Earlier, fluorescent protein‐based biosensors were reported with static quenching, energy transfer between chromophore and colored metal ion and altered protein structure 9,10,19‐25 . In this study, the confirmation of protein with novel chemical amine moiety allowed us to evaluate the fluorescent protein‐based reagent‐less metal sensing system.…”

“…Earlier, fluorescent protein-based biosensors were reported with static quenching, energy transfer between chromophore and colored metal ion and altered protein structure. 9,10,[19][20][21][22][23][24][25] In this study, the confirmation of protein with novel chemical amine moiety allowed us to evaluate the fluorescent protein-based reagent-less metal sensing system. The metal sensing ability of the protein was determined by analyzing the change in fluorescence of amGFP upon treatment with various metal ions.…”

“…Frequently, two experimental approaches, namely reassignment of sense (residue-specific incorporation) and nonsense suppression (site-specific incorporation), are used for in vivo incorporation of NCAAs into recombinant proteins. [17][18][19][20][21][22][23][24][25] In the work reported here, we aimed to introduce donor-acceptor non-natural chemistry in the GFP chromophore, offering significant Stokes shifts to GFP. 11,26 We aimed to incorporate aminotyrosine as an electron-donating group (amine group) into the GFP chromophore for the development of an efficient red-shifted fluorescent protein-based biosensor that can also act as a bio-cleaner for the removal of Cu 2+ ions from environmental samples.…”

“…Synthetic NCAAs offer non‐natural potential with new functional chemistry for proteins with enhanced spectral properties. Frequently, two experimental approaches, namely reassignment of sense (residue‐specific incorporation) and nonsense suppression (site‐specific incorporation), are used for in vivo incorporation of NCAAs into recombinant proteins 17‐25 . In the work reported here, we aimed to introduce donor–acceptor non‐natural chemistry in the GFP chromophore, offering significant Stokes shifts to GFP 11,26 .…”

Stabilization of Salivary Biomarkers

“…Sol–gel encapsulation of labile molecules within a silica matrix has been extensively explored to enhance protein thermal stability. The chemical reactions involved in sol–gel encapsulation are based on the hydrolysis of alkoxide precursors under acidic or basic conditions, followed by condensation and polycondensation of the hydroxylated units, which lead to the formation of a porous gel. − Protein content can be simultaneously entrapped in these porous, optically transparent silica matrices, which hinders proteins’ rotational freedom, thus preserving their native structure and avoiding aggregation, although protein unfolding can occur due to water encapsulation within the pores . Despite the complexity and time-consuming reaction steps, silica matrixes are compatible with different analytical techniques and sol–gel entrapment has been extensively used to increase the stability of proteins in sensing devices and drug delivery systems. , More recently, sol–gel chemistries have been applied to coat and protect DNA molecules against the action of DNases and temperature-induced degradation opening novel routes toward the stabilization of genomic content in biological fluids.…”