Title
Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States
Author
Marialaura Marchetti, Luca Ronda, Riccardo Percudani, Stefano Bettati
Year
2020
Journal
Sensors
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.
Full Article
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Protein folding, Protein stability, Kinetics, Stereochemistry, Sensors, Materials, Biochemistry