Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples

Abstract

Serum uric acid (SUA) is an important biomarker for prognosis and management of gout and other diseases. The development of a low-cost, simple, rapid and reliable assay for SUA detection is of great importance. In the present study, to save the cost of enzyme production and to shorten the reaction time for uric acid quantification, bifunctional proteins with uricase and peroxidase activities were engineered. In-frame fusion of Candida utilis uricase (CUOX) and Vitreoscilla hemoglobin (VHb) resulted in two versions of the bifunctional protein, CUOX-VHb (CV) and VHb-CUOX (VC). To our knowledge, this is the first report to describe the production of proteins with uricase and peroxidase activities. Based on the measurement of the initial rates of the coupled reaction (between uricase and peroxidase), CV was proven to be the most efficient enzyme followed by VC and native enzymes (CUOX+VHb), respectively. CV was further applied for the development of an assay for colorimetric detection of SUA, which was based on VHb-catalyzed oxidation of Amplex Red in the presence of hydrogen peroxide (H2O2). Under the optimized conditions, the assay exhibited a linear relationship between the absorbance and UA concentration over the range of 2.5 to 50 μM, with a detection limit of 1 μM. In addition, the assay can be performed at a single pH (8.0) so adjustment of the pH for peroxidase activity was not required. This advantage helped to further reduce costs and time. The developed assay was also successfully applied to detect UA in pooled human serum with the recoveries over 94.8%. These results suggest that the proposed assay holds great potential for clinical application.

Colorimetric and electrochemical dual-mode uric acid determination utilizing peroxidase-mimicking activity of CoCu bimetallic nanoclusters

“…9 Phuadraksa et al investigated the use of uricase and Vitreoscilla hemoglobin by forming a bifunctional protein in the colorimetric detection of UA with a short reaction time. 10 Lu et al developed a colorimetric sensor for the detection of UA using Cu 2+ to catalyze the TMB–H 2 O 2 system and UA to generate H 2 O 2 through uricase. 11 However, there are still some deficiencies in the detection of UA by colorimetric methods.…”

Ultrasensitive and selective colorimetric detection of uric acid using peroxidase mimetic activity of biogenic palladium nanoparticles

“…[ 3 ] The clinical approaches for uric acid concentration assessment are based on the examination of serum uric acid from 2.4–6.0 mg/dl (143–357 μM) in adult women and 3.4–7.0 mg/dl (202–416 μM) in adult men. [ 4 ] The condition in which there are elevated concentrations of uric acid in serum of more than 6.8 mg/dl (405 μM) is known as hyperuricemia. Therefore, the measurement of uric acid concentrations in the serum plays a significant role in the early diagnosis and treatment of relevant diseases.…”