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.
Highlights
Oxidative stress is defined as an imbalance between the presence of reactive species and the antioxidant body response
The attention is progressively focusing on allantoin, a stable and polar small molecule generated in humans from the non-enzymatic oxidation of uric acid, as a potential biomarker for routine clinical analyses
The fluorescence values obtained from the plate reading were subtracted of the blank and normalized, and their dependence on allantoin concentration was fitted to a linear equation (Figure 5)
Summary
Oxidative stress is defined as an imbalance between the presence of reactive species and the antioxidant body response. The relevance of a biosensor resides in the selectivity and sensitivity in target quantification and reusability, determining its applicability as a tool for routine clinical practice With this purpose, we entrapped puuE in a wet nanoporous silica gel matrix, a strategy largely exploited in enzyme immobilization because of its several advantages, among which the easy and flexible chemistry, with mild chemical and physical entrapment conditions compatible with protein stability, the optical transparency allowing signal detection with conventional spectroscopic techniques, the ease of separation of the active material from the reaction solution [24,25,26,27,28,29,30,31]. The presented results demonstrate the applicability of this strategy to a chemical/enzymatic assay for allantoin quantification [22], in which respect it represents a major improvement in terms of reusability and storability
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