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Uric acid (UA) serves as an important biochemical marker of various diseases, making the development of a novel method for its rapid and straightforward visual detection highly valuable. In this study, a uricase-based cellulose membrane biosensor (UCMB) was constructed by immobilizing uricase via a Schiff base reaction and nitroblue tetrazolium chloride (NBT) through adsorption. The UCMB detects UA through a mechanism in which uricase catalyzes the oxidation of UA, generation O 2 -· radicals that subsequently oxidize NBT to formazan, producing a distinctive color change from yellow to purple. The UCMB demonstrated successful visual detection of UA within 15 min, allowing for rapid naked-eye analysis. Additionally, the biosensor quantitatively detected UA over a broad linear range from 0 to 1000 μM, with a low detection limit of 3.88 μM. Most notably, the UCMB has accurately measured UA in human serum samples, comparable to the results from a commercial UA meter. These findings suggest that the UCMB can serve as a simple and reliable tool for early diagnosis of UA-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gout, marked by the deposition of sodium urate crystals in joints and peripheral tissues, presents a considerable health challenge. Recent research has shown a growing interest in nanozyme-based treatments for gout. However, literature on nanozymes that combine uricase-like (UOX) activity for uric acid (UA) degradation with catalase (CAT)-like activity for H 2 O 2 elimination through a self-cascade reaction is limited. Herein, we discovered that two-dimensional Pd@Ir nanosheets (NSs) exhibit UOX and CAT activities effectively. Notably, we observed a size-dependent effect of Pd@Ir on activation energy during UA degradation, with the larger Pd@Ir NSs demonstrating a lower energy barrier. The 46-nm Pd@Ir had activation energy as low as 35.9 kJ/mol, surpassing the efficiency of natural bacterial uricase and most reported nanozymes. Through a tandem self-cascade reaction of Pd@Ir, UA was effectively degraded via UOX activity, while the byproduct H 2 O 2 was simultaneously eliminated by CAT-like activity. Cell experiments revealed that Pd@Ir protect normal cells from oxidative stress and promote cell proliferation, demonstrating an excellent self-cascade effect. Additionally, Pd@Ir substantially alleviated gout symptoms in monosodium urate-induced acute gout mice without causing toxic effects on biological organs and tissues. This study opens new avenues for using nanozyme-based cascade reaction systems in the treatment of metabolic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microbial uricase is an essential enzyme in purine degradation and the development of low-purine food. High enzyme activity and an appropriate optimum pH must be established for low-purine food. Uricases from Neurospora crassa , Streptomyces coelicolor , Alkalihalobacillus clausii , Bacillus subtilis , and Brevibacterium casei were heterologously expressed in Escherichia coli . Uricase from Alkalihalobacillus clausii (AC-PUCL) showed the most potent enzyme activity (249.19 IU/mL) at 37 °C and pH 7.0. This is close to the pH of plant-based food. The K m and K m / K cat values of AC-PUCL were 30.12 μM and 1.46 s -1 μM -1 , respectively. Furthermore, the crystal structures of uricases from different sources revealed that hydrogen bonds could enhance substrate affinity and strong enzyme activity. In addition, the high enzyme activity may be contributed by the active pockets with an appropriate size. Finally, AC-PUCL helped reduce the purine substances in soybean, pea, and kidney bean, with residual uric acid can not be detected at pH 8.6, suggesting a promising industrial application.

A Cr-doped VO 2 nanobelt (Cr/VO 2 ) with remarkable peroxidase-like activity was synthesized and coupled with uricase to catalyze the cascade reaction for  detection of uric acid. Notably, the affinity of Cr/VO 2 for 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate (TMB) and hydrogen peroxide (H 2 O 2 ) is tenfold and 20-fold higher, respectively, than that of horseradish peroxidase (HRP). The Cr/VO 2 exhibits highly reactive and stable peroxidase activity at temperatures of 20-60 ℃. Thus, the reaction catalyzed by Cr/VO 2 can be carried out at room temperature, greatly simplifying the testing process. This enzyme cascade nanozyme based colorimetric assay enables the selective and simple detection of uric acid within 30 min with a detection limit of only 0.34 μM, and has an observable potential in clinical applications., Competing Interests: Declarations. Ethical approval: Not applicable. Competing interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A sensor for uric acid (UA) based on the urate oxidase enzyme (UOx) immobilized in novel Co-based aerogels with transition metals synthesized by the sol-gel method was developed and evaluated. The Co-based aerogels: Co, Ni-Co and Pd-Co were physicochemically characterized by XRD and HR-TEM. The surface area values of 53, 57 and 66 m 2 g-1 were determined for Co, Ni-Co and Pd-Co, respectively by N 2 adsorption-desorption technique. Co-based aerogels were mixed by cross-linking with UOx enzymes and electrochemically characterized in buffers at pH 7.4 and 5.6 (pH values reported for biological fluids such as blood and sweat) in the presence of different uric acid concentrations. Co-based aerogels with UOx showed improved performance as a uric acid biosensor compared to using the enzyme alone. At a pH of 7.4, a higher sensitivity of 11 μA μM -1 was obtained with Pd-Co/UOx, 1.6 times higher than with UOx. At a pH value of 5.6, the highest sensitivity is achieved with Ni-Co/UOx. Stability and selectivity tests were performed in the presence of biological interferents without significant changes in the sensor. These results indicate a pleasing synergistic activity between Co-based aerogels and the enzyme., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Ledesma-Garcia reports financial support was provided by Mexican Council for Humanities, Sciences and Technologies., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metabolic processes in living organisms depend on the synergistic actions of enzymes working in proximity and in concert, catalyzing reactions effectively while regulating the formation of metabolites. This enzyme synergy offers promising therapeutic application for diseases such as alcohol intoxication, cancer, and hyperinflammation. Despite their potential, the clinical translation of enzyme cascades is restricted by challenges including poor enzyme stability, short half-life, and a lack of delivery strategies that maintain enzyme proximity. In this study, multi-enzyme nanocascades synthesized are developed through in situ atom transfer radical polymerization using a zwitterionic monomer. This method markedly enhances enzyme stability and proximity, thereby prolonging their circulation half-life after systemic administration. It is demonstrated that the nanocascades of uricase and catalase effectively reduce uric acid levels without excessive hydrogen peroxide production, providing a potential antidote for hyperuricemia. Moreover, in a murine breast cancer model, the nanocascades of glucose oxidase and catalase inhibited tumor progression and enhanced the therapeutic efficacy of doxorubicin. The prolonged circulation and promoted reaction efficacy of these nanocascades underscore their substantial potential in enzyme replacement therapy and the treatment of various diseases., (© 2024 Wiley‐VCH GmbH.)

Despite that the current anti-hyperuricemia drugs can effectively reduce uric acid (UA) levels, imprecise medication dosage or uncontrolled lowering of UA levels may result in undesired effects. To address this issue, a closed-loop cascade strategy based on a biocompatible network composite, NW-FPNP/uricase (UOX), is proposed for on-demand regulation of UA levels. NW-FPNP/UOX is constructed by encapsulation of UOX) as UA-responsive element and FPNP, a nanoparticle of phenylboronic acid modified xanthine oxidase (XOD) inhibitor febuxostat, as H 2 O 2 -sensitive element with AMP/Gd 3+ network. It interrelates the UA metabolization and generation processes into a closed loop of cascade reactions involving UOX-catalyzed UA metabolization and H 2 O 2 generation, H 2 O 2 -triggered febuxostat regeneration and XOD inhibition, and XOD-catalyzed UA generation. Through UA level-dependent auto-adjustment of XOD activity, specially 6% at 600 × 10 -6 m UA compared to 82% at 100 × 10 -6 m, UA levels can be regulated to an appropriate range through dynamically balancing UA metabolization and generation. This biocompatible on-demand UA regulation system prevents the overdose of UA-lowering medications and avoids hypouricemia in hyperuricemia treatment, demonstrating great potential in intelligent UA level management. This work also introduces a new concept of a closed-loop cascade strategy for on-demand regulation of biochemical indicators within specific thresholds., (© 2024 Wiley‐VCH GmbH.)

A pasting-3D microfluidic paper-based analytical device (P-3D μPAD) was developed. It enabled an efficient cascade reaction between urate oxidase (UOX) and Fe/Pt-doped carbon nanoparticles (Fe/Pt-CNPs) for visual colorimetric detection of uric acid (UA). The novel Fe/Pt-CNP nanozyme performed high peroxidase-like activity toward 3,3',5,5'-tetramethylbenzidine (TMB) and H 2 O 2 with Michaelis - Menten constants (K m ) of 0.97 and 2.30 mM, respectively. The UOX-Fe/Pt-CNP system was incorporated into P-3D μPAD: the 1st layer was UOX hydrolysis reaction under alkaline pH, and the 2nd layer was Fe/Pt-CNPs catalyzing H 2 O 2 to oxidize TMB at acidic pH. The separated two layers allowed cascade reaction under different working pH without sacrificing the activity of UOX or Fe/Pt-CNPs. The images were captured and analyzed by the camera and "Color Recognition" application using a  smartphone. The linear range of P-3D μPAD for UA was 25-1000 μM with a limit of detection of 10 μM which met the requirements for clinical applications. The high accuracy of P-3D μPAD for UA detection in invasive (blood) and noninvasive (saliva) samples has been confirmed by the biochemical analyzer. This work offers a sensitive, flexible, affordable, and disposable tool for on-site UA level monitoring and provides new insight into natural enzyme and nanozyme tandem systems for biosensing., Competing Interests: Declarations. Ethics approval: All experiments were with permission from the Ethic Committees of Shanxi Bethune Hospital (no. SBQLL-2023–010), and the approval date is March 8, 2023. All volunteers signed documents providing their informed consent, and all methods were according to the relevant guidelines and institutional regulations. Competing interest: The autho s declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A novel dual-parameter optical fiber biosensor based on surface plasmon resonance (SPR) for simultaneous measurement of urea and uric acid concentrations is proposed in this paper. Based on the principle of positive and negative electric combination, ZnO nanoparticles is selected as the matrix for immobilizing urease and uricase with selective recognition ability, which can also be used as a sensitizing material to increase the refractive index detection sensitivity of SPR by 22%. Then, Nafion ion exchange membrane was introduced to wrap the urea sensing area to avoid crosstalk caused by the overlap of adjacent sensing areas. The difference in the number of enzymes loaded on ZnO nanoparticles was used to meet the refractive index gap for forming dual SPR detection channels. Experiments indicate that the sensitivity of urea detection is 1.6 nm/mM in the range of 1-9 mM urea concentration. The detection sensitivity of uric acid is 36 nm/mM in the concentration range of 50-500 μM. The detection ability of the sensor to actual biological samples was verified by serum samples, which proved that the sensor has practical application potential and reliable selectivity. It can provide a new idea for the research and development of multi-parameter optical fiber biosensors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this article, we present the first demonstration of a distance-based paper analytical device (dPAD) for uric acid quantification in human urine samples with instrument-free readout and user-friendliness for the rapid diagnosis and prognosis of various related diseases. By employing gold nanoparticles (AuNPs) as a peroxidase-like nanozyme, our proposed technique eliminates the utilization of horseradish peroxidase (HRP), making the device cost-effective and stable. In our dPAD, uric acid in the sample is oxidized by the uricase enzyme and subsequently catalysed with AuNPs in the sample zone, generating hydroxyl radicals (˙OH). Then, the produced ˙OH reacts with 3,3'-diaminobenzidine (DAB) to form poly DAB (oxDAB), resulting in a coloured distance signal in the detection zone of the dPAD. The variation of the distance of the observed red-brown colour is directly proportional to the uric acid concentration. Our sensor exhibited a linear range from 0.50 to 6.0 mmol L -1 ( R 2 = 0.9922) with a detection limit (LOD) of 0.25 mmol L -1 , covering the clinical range of uric acid in urine. Hence, there is no need for additional sample preparation or dilution. Additionally, this assay is highly selective, with no interferences. We also found that this approach could accurately and precisely determine uric acid in human control samples with the recovery ranging from 99.37 to 100.35 with the highest RSD of 4.05%. Our method is comparable with the use of a commercially available uric acid sensor at a 95% confidence interval. Consequently, the developed dPAD offers numerous advantages such as cost-effectiveness, simplicity, and ease of operation with unskilled individuals. Furthermore, this concept can be applied for extensive biosensing applications in monitoring other biomarkers as an alternative analytical point-of-care (POC) device.

Gouty arthritis is a chronic and progressive disease characterized by high urate levels in the joints and by an inflammatory immune microenvironment. Clinical data indicate that urate reduction therapy or anti-inflammatory therapy alone often fails to deliver satisfactory outcomes. Here we have developed a smart biomimetic nanosystem featuring a 'shell' composed of a fusion membrane derived from M2 macrophages and exosomes, which encapsulates liposomes loaded with a combination of uricase, platinum-in-hyaluronan/polydopamine nanozyme and resveratrol. The nanosystem targets inflamed joints and promotes the accumulation of anti-inflammatory macrophages locally, while the uricase and the nanozyme reduce the levels of urate within the joints. Additionally, site-directed near-infrared irradiation provides localized mild thermotherapy through the action of platinum and polydopamine, initiating heat-induced tissue repair. Combined use of these components synergistically enhances overall outcomes, resulting in faster recovery of the damaged joint tissue., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL) -1 , and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Stimulated by informal conversations at the XVII International Small Angle Scattering (SAS) conference (Traverse City, 2017), an international team of experts undertook a round-robin exercise to produce a large dataset from proteins under standard solution conditions. These data were used to generate consensus SAS profiles for xylose isomerase, urate oxidase, xylanase, lysozyme and ribonuclease A. Here, we apply a new protocol using maximum likelihood with a larger number of the contributed datasets to generate improved consensus profiles. We investigate the fits of these profiles to predicted profiles from atomic coordinates that incorporate different models to account for the contribution to the scattering of water molecules of hydration surrounding proteins in solution. Programs using an implicit, shell-type hydration layer generally optimize fits to experimental data with the aid of two parameters that adjust the volume of the bulk solvent excluded by the protein and the contrast of the hydration layer. For these models, we found the error-weighted residual differences between the model and the experiment generally reflected the subsidiary maxima and minima in the consensus profiles that are determined by the size of the protein plus the hydration layer. By comparison, all-atom solute and solvent molecular dynamics (MD) simulations are without the benefit of adjustable parameters and, nonetheless, they yielded at least equally good fits with residual differences that are less reflective of the structure in the consensus profile. Further, where MD simulations accounted for the precise solvent composition of the experiment, specifically the inclusion of ions, the modelled radius of gyration values were significantly closer to the experiment. The power of adjustable parameters to mask real differences between a model and the structure present in solution is demonstrated by the results for the conformationally dynamic ribonuclease A and calculations with pseudo-experimental data. This study shows that, while methods invoking an implicit hydration layer have the unequivocal advantage of speed, care is needed to understand the influence of the adjustable parameters. All-atom solute and solvent MD simulations are slower but are less susceptible to false positives, and can account for thermal fluctuations in atomic positions, and more accurately represent the water molecules of hydration that contribute to the scattering profile., (open access.)

With the demand for mass production of protein drugs, solubility has become a serious issue. Extrinsic and intrinsic factors both affect this property. A homotetrameric cofactor-free urate oxidase (UOX) is not sufficiently soluble. To engineer UOX for optimum solubility, it is important to identify the most effective factor that influences solubility. The most effective feature to target for protein engineering was determined by measuring various solubility-related factors of UOX. A large library of homologous sequences was obtained from the databases. The data was reduced to six enzymes from different organisms. On the basis of various sequence- and structure-derived elements, the most and the least soluble enzymes were defined. To determine the best protein engineering target for modification, features of the most and least soluble enzymes were compared. Metabacillus fastidiosus UOX was the most soluble enzyme, while Agrobacterium globiformis UOX was the least soluble. According to the comparison-constant method, positive surface patches caused by arginine residue distribution are appropriate targets for modification. Two Arg to Ala mutations were introduced to the least soluble enzyme to test this hypothesis. These mutations significantly enhanced the mutant's solubility. While different algorithms produced conflicting results, it was difficult to determine which proteins were most and least soluble. Solubility prediction requires multiple algorithms based on these controversies. Protein surfaces should be investigated regionally rather than globally, and both sequence and structural data should be considered. Several other biotechnological products could be engineered using the data reduction and comparison-constant methods used in this study., Competing Interests: Declarations. Ethics Approval: The bioinformatic nature of our study did not involve human or animal subjects; thus, ethical approval was not applicable. Consent to Participate and Consent to Publish: All authors consent to participate and publish the paper in Applied Biochemistry and Biotechnology. Conflict of Interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The uric acid (UA) level is an important physiological indicator of the human body, and its abnormality can lead to a series of diseases. Therefore, the immediate detection of uric acid concentration has broad application prospects. Commonly used methods for the analysis of uric acid include chromatography, high-performance capillary electrophoresis and electrochemical methods. However, these methods have the disadvantages of cumbersome sample pre-treatment, high cost, time-consuming, and the need for experimental instruments and professional operators, which are extremely unfavorable for the detection of uric acid and the diagnosis of related diseases in resource-limited areas. In this study, a portable visualization method was developed for the detection of uric acid using hydrogen peroxide (H 2 O 2 ) test strips. Uric acid enzyme specifically catalyzes the oxidation of uric acid to produce H 2 O 2 , which causes a significant change in the color of the H 2 O 2 test strip. The response has good linearity in the range of 1 ∼ 50 μg mL -1 . Thus, it provides a simple, rapid, and cost-effective visualized bioassay for uric acid.

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu 2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu 2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 μM) with a low limit (4.4 μM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An enzymatic amperometric uric acid (UA) biosensor was successfully developed by modifying a screen-printed carbon electrode (SPCE) with Prussian blue-poly(3,4-ethylene dioxythiophene) polystyrene sulfonate composite (PB-PEDOT:PSS). The modified SPCE was coated with gold nanoparticles-graphene oxide-chitosan composite cryogel (AuNPs-GO-CS cry). Uricase (UOx) was directly immobilized via chemisorption on AuNPs. The nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. The electrochemical characterization of the modified electrode was performed by cyclic voltammetry and electrochemical impedance spectroscopy. UA was determined using amperometric detection based on the reduction current of PB which was correlated with the amount of H 2 O 2 produced during the enzymatic reaction. Under optimal conditions, the fabricated UA biosensor in a flow injection analysis (FIA) system produced a linear range from 5.0 to 300 μmol L -1 with a detection limit of 1.88 μmol L -1 . The proposed sensor was stable for up to 221 cycles of detection and analysis was rapid (2 min), with good reproducibility (RSDs < 2.90 %, n = 6), negligible interferences, and recoveries from 94.0 ± 3.9 to 101.1 ± 2.6 %. The results of UA detection in blood plasma were in agreement with the enzymatic colorimetric method (P > 0.05)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metal nanozymes have offered attractive opportunities for biocatalysis and biomedicine. However, fabricating nanozymes simultaneously possessing highly catalytic selectivity and activity remains a great challenge due to the lack of three-dimensional (3D) architecture of the catalytic pocket in natural enzymes. Here, we integrate rhodium nanocluster (RhNC), reduced graphene oxide (rGO), and protamine (PRTM, a typical arginine-rich peptide) into a composite facilely based on the single peptide. Remarkably, the PRTM-RhNC@rGO composite displays outstanding selectivity, activity, and stability for the catalytic degradation of uric acid. The reaction rate constant of the uric acid oxidation catalyzed by the PRTM-RhNC@rGO composite is about 1.88 × 10 -3 s -1 (4 μg/mL), which is 37.6 times higher than that of reported RhNP ( k = 5 × 10 -5 s -1 , 20 μg/mL). Enzyme kinetic studies reveal that the PRTM-RhNC@rGO composite exhibits a similar affinity for uric acid as natural uricase. Furthermore, the uricase-like activity of PRTM-RhNC@rGO nanozymes remains in the presence of sulfur substances and halide ions, displaying incredibly well antipoisoning abilities. The analysis of the structure-function relationship indicates the PRTM-RhNC@rGO composite features the substrate binding site near the catalytic site in a confined space contributed by 2D rGO and PRTM, resulting in the high-performance of the composite nanozyme. Based on the outstanding uricase-like activity and the interaction of PRTM and uric acid, the PRTM-RhNC@rGO composite can retard the urate crystallization significantly. The present work provides new insights into the design of metal nanozymes with suitable binding sites near catalytic sites by mimicking pocket-like structures in natural enzymes based on simple peptides, conducing to broadening the practical application of high-performance nanozymes in biomedical fields.

In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O 2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes., (© 2024. The Author(s).)

The effect of strong metal-support interaction (SMSI) has never been systematically studied in the field of nanozyme-based catalysis before. Herein, by coupling two different Pd crystal facets with MnO 2 , i.e., (100) by Pd cube (Pd c ) and (111) by Pd icosahedron (Pd i ), we observed the reconstruction of Pd atomic structure within the Pd-MnO 2 interface, with the reconstructed Pd c (100) facet more disordered than Pd i (111), verifying the existence of SMSI in such coupled system. The rearranged Pd atoms in the interface resulted in enhanced uricase-like catalytic activity, with Pd c @MnO 2 demonstrating the best catalytic performance. Theoretical calculations suggested that a more disordered Pd interface led to stronger interactions with intermediates during the uricolytic process. In vitro cell experiments and in vivo therapy results demonstrated excellent biocompatibility, therapeutic effect, and biosafety for their potential hyperuricemia treatment. Our work provides a brand-new perspective for the design of highly efficient uricase-mimic catalysts.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC 50 of 5.34 ± 0.65 μM, which is less potent than benzbromarone (2.01 ± 0.36 μM) but more potent than lesinurad (10.36 ± 1.23 μM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC 50 of 1.04 ± 0.23 μM. Its metabolic product, gallic acid, inhibited XOD with an IC 50 of 0.91 ± 0.14 μM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t 1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett-Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropyl β-d -1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD 600 ) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml -1 and a final OD 600 of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production., (© 2024 IOP Publishing Ltd.)

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N 3 -ZBP) using a " grafting-from " strategy involving RAFT and ATRP methods was synthesized, and a trans -cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10 -2  μg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer., (© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework-8 (mp20@ZIF-8) followed by the modification on rGO-screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate-mp20 interaction., (© 2023 Wiley Periodicals LLC.)

In this work, palm oil fiber (POF) grafted functionalized multiwall carbon nanotube (FMWCNT) decorated ferrocene (Fc) has been drop coated on a platinum electrode (Pt), in which uricase (UOx) has been chemically immobilized for sensitive and selective biosensing of uric acid (UA). Through the use of EDC/NHS, a stable bioelectrode (UOx/Fc/FMWCNT-POF/Pt) was obtained and characterized by FTIR/ATRIR, XRD, Raman, EA/EDX, TGA, SEM, TEM, CV, EIS, CA, and DPV. Results from DPV showed the rapid response of the developed bioelectrode towards UA (0.185 V) with high sensitivity (41.14 μA mM -1 ) and good limit of detection (19 μM) in the linear range 10-1000 μM. The low value of Michaelis-Menten constant ( k m = 31.364 μM) shows high affinity of the UA towards the enzyme at the electrode surface. The developed biosensor demonstrates good reproducibility, repeatability, and stability with a deviation of less than 2.5%, and was successfully applied for human blood sample analysis. The CA study revealed a fast response time (2 s) of the sensor. The work has pioneered a new addition to the class of tailorable chemical species for biosensor development and proven to be a promising new tool for point of care testing (POCT) applications.

The synthesis of ultrasmall Ta 2 O 5 nanoparticle anchored Pt atom using aspartic acid-functionalized graphene quantum dot (Asp-GQD) is reported. The Asp-GQD was combined with tantalic acid and chloroplatinic acid to rapidly form water-soluble Ta-Asp-GQD and Pt-Asp-GQD complex. Followed by thermal annealing at 900 °C in N 2 to obtain Ta 2 O 5 -Asp-GQD-Pt. The study shows that the introduction of Asp-GQD as a chelating agent and p-type semiconductor achieves to the formation of ultrasmall Ta 2 O 5 nanoparticle, PN junction at the interface and Pt single atom anchored on the surface of Ta 2 O 5 nanocrystals. The unique structure realizes ultrahigh uricase activity and catalase activities of Ta 2 O 5 -Asp-GQD-Pt. The Ta 2 O 5 -Asp-GQD-Pt was used as the bifunctional sensing material for the construction of an electrochemical uric acid sensor. The differential pulse voltammetric current at 0.45 V linearly increases with the increase of uric acid concentration in the range 0.001-5.00 mM with the detection limit of 0.41 μM (S/N = 3). The sensor exhibits a much better sensitivity compared with the reported methods for the detection of uric acid. The proposed analytical method has been applied to the electrochemical detection of uric acid in human serum with a spiked recovery of 95-105%. The study also offers one way to design and synthesize multifunctional sensing materials with high catalytic activity., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cascade reactions catalyzed by natural uricase and mimic peroxidase (MPOD) have been applied for uric acid (UA) detection. However, the optimal catalytic activity of MPOD is mostly in acidic conditions (pH 2-5), mismatching the optimal catalytic alkaline environment of uricase. In this paper, using CuSO 4 and urea as raw materials, a MPOD with high catalytic activity in alkaline environment was synthesized by hydrothermal method. Then, based on coupling reaction of uricase/UA/MPOD/guaiacol (GA) system, a novel spectrophotometric method was established to detect 5-60 μmol/L UA (limit of detection = 3.14 μmol/L (S/N = 3)) and accurately quantified serum UA (275.6 ± 39.9 μmol/L, n = 5) with 95-105% of standard addition recovery. The results were consistent with commercial UA kit (p > 0.05). The MPOD could replace natural POD to reduce the cost of UA detection due to simple preparation and cheap raw materials, and is expected to achieve the specific detection of some substances, like glucose and cholesterol, combined with glucose oxidase and cholesterol oxidase., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose. All the experiments related to human serum were approved by the medical ethics committee of Sichuan University, and performed in accordance with the guidelines and ethical standards of our lab, and informed consent was obtained from all the participants., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A novel uricase producing marine bacterium Priestia flexa alkaAU was isolated and identified. The 16S rDNA and the uricase coding gene were sequenced, analyzed and submitted to GenBank. The uricase from Priestia flexa alkaAU (PFU) was purified, determined to be 58.87 kDa, and conjugated with carboxymethyl chitosan (CMCS) by ionic gelation. CMCS conjugation had no effect on the optimum pH of PFU but decreased the optimum temperature by 10 °C. CMCS conjugation increased the specific activity of PFU by 53% at the human body temperature (37 °C) and small intestine's pH (pH 6.8). Uricase thermostabilizing ability of CMCS was significant in the range of 37-80 °C but not at lower temperatures. For improvement of the pH stability of PFU, CMCS was more effective at pHs 3-5 than pHs 6-11. CMCS increased the half-life of PFU against artificial intestinal fluid by 1.5 folds, which demonstrated the potential capability of CMCS-PFU for oral administration.

In a new approach, we considered the special affinity between Ni and poly-histidine tags of recombinant urate oxidase to utilize Ni-MOF for immobilizing the enzyme. In this study, a carbon paste electrode (CPE) was modified by histidine-tailed urate oxidase (H-UOX) and nickel-metal-organic framework (Ni-MOF) to construct H-UOX/Ni-MOF/CPE, which is a rapid, sensitive, and simple electrochemical biosensor for UA detection. The use of carboxy-terminal histidine-tailed urate oxidase in the construction of the electrode allows the urate oxidase enzyme to be positioned correctly in the electrode. This, in turn, enhances the efficiency of the biosensor. Characterization was carried out by X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), Brunauer-Emmett-Teller (BET), and field emission scanning electron microscopy (FE-SEM). At optimum conditions, the biosensor provided a short response time, linear response within 0.3-10 µM and 10-140 µM for UA with a detection limit of 0.084 µM, repeatability of 3.06%, and reproducibility of 4.9%. Furthermore, the biosensor revealed acceptable stability and selectivity of UA detection in the presence of the commonly coexisted ascorbic acid, dopamine, L-cysteine, urea, and glucose. The detection potential was at 0.4 V vs. Ag/AgCl., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The abnormal levels of uric acid (UA) in body fluids are associated with gout, type (II) diabetes, leukemia, Lesch-Nyhan syndrome, uremia, kidney damage, and cardiovascular diseases. Also, the presence of uricase (UOx) symbolizes genetic disorders and corresponding complications. Therefore, the detection of UA and UOx in the body fluids is significant for clinical diagnosis. 4-Cyano-4'-pentylbiphenyl (5CB, a nematic liquid crystal (LC)) was doped with octadecyl trimethylammonium bromide (OTAB, a cationic surfactant), which formed a self-assembled monolayer at the aqueous/5CB interface. The UOx-catalyzed oxidation of UA yielded H 2 O 2 , releasing the single-strand deoxyribonucleic acid (ssDNA) from the nanoceria/ssDNA complex. The interaction of the released ssDNA with OTAB disrupted the monolayer at the aqueous/5CB interface, which resulted in a dark to bright change when observed through a polarized optical microscope. The LC-based sensor allowed the detection of UA with a linear range of 0.01-10 μM and a limit of detection (LOD) of 0.001 μM. The UA detection was also performed in human urine samples and the results were comparable to that of a standard commercial colorimetric method. Similarly, the detection of UOx was performed, with a noted linear range of 20-140 μg/mL. The LOD was as low as 0.34 μg/mL. The detection of UOx was also demonstrated in human serum samples with excellent performance. This method provides a robust sensing platform for the detection of UA and UOx and has potential for applications in clinical analysis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this work, a novel multifunctional nano-enzyme platform was developed and used for enzymatic and ratiometric electrochemical biosensing of uric acid (UA). Boron nanosheets (BNSs) were prepared through ultrasound-assisted liquid-phase exfoliation, followed by the loading of doxorubicin (DOX) to form BNSs-DOX complex. The complex was drop-casted on glassy carbon electrode (GCE) surface to prepare BNSs-DOX/GCE. Cobalt-based metal-organic framework (MOF) with encapsulation of urate oxidase (UOx) was in-situ copolymerized and electrodeposited on the BNSs-DOX surface to construct UOx@MOF/BNSs-DOX nanohybrid-modified GCE. The modified electrode serves as an artificial nano-enzyme sensing platform and presents multifunctional functions, including DOX-loaded BNSs carrier, UOx-enzyme immobilization, enzymatic redox and ratiometric electrochemical sensing of UA. The platform was explored as a new ratiometric electrochemical biosensor to detect UA in the concentration range of 0.1-200 μM, with a low limit of detection of 0.025 μM. Experimental results testify high selectivity, sensitivity and stability toward efficient detection of UA over potential interferents, revealing high detection accuracy and repeatability. The explored biosensor shows superior detection performances in real biological samples, together with high detection recoveries. Excellent properties and functions endow the biosensor with great prospects for precise screening and early diagnosis of UA-relevant malignant diseases in clinic., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Urate oxidase (EC 1.7.3.3) is a key enzyme in the purine metabolism which is applied in the treatment of gout and also, as a diagnostic reagent for uric acid detection. In the current study, the trehalose (TRE) effects as an additive on the structural stability and function of uricase were investigated. For recombinant expression of UOX in E. coli BL21 cells, firstly the coding sequence was subcloned into the pET-28a vector and after induction with IPTG, the recombinant UOX was purified by affinity chromatography using a Ni-NTA agarose column. To specify the trehalose effects on the urate oxidase (UOX) structure, optimum pH, optimum temperature, kinetic and thermodynamic parameters and also, the intrinsic fluorescence of UOX in the absence and presence of trehalose were examined. The UOX half-life is 24.32 min at 40 °C, whereas the UOX-TRE has a higher half-life (32.09 min) at this temperature. Generally, our findings confirm that trehalose has a protective effect on the enzyme structure. Optimum pH and temperature were 9 and 25 °C, respectively for both the naked and treated enzymes and their activity retained 42.18 and 64.80%, respectively after 48 h of incubation at room temperature. Also, theoretical results indicate that the random coil of the enzyme was converted to α-helix and β-sheet in the presence of trehalose which may preserve the integrity of the active site of the enzyme and increased the enzymatic activity. The MD simulation results indicated greater stability of the uricase structure in the presence of trehalose.Communicated by Ramaswamy H. Sarma.

The stability of the tetrameric enzyme urate oxidase in complex with excess of 8-azaxanthine was investigated either under high hydrostatic pressure per se or under a high pressure of argon. The active site is located at the interface of two subunits, and the catalytic activity is directly related to the integrity of the tetramer. This study demonstrates that applying pressure to a protein-ligand complex drives the thermodynamic equilibrium towards ligand saturation of the complex, revealing a new binding site. A transient dimeric intermediate that occurs during the pressure-induced dissociation process was characterized under argon pressure and excited substates of the enzyme that occur during the catalytic cycle can be trapped by pressure. Comparison of the different structures under pressure infers an allosteric role of the internal hydrophobic cavity in which argon is bound, since this cavity provides the necessary flexibility for the active site to function.

We report enzyme-powered upconversion-nanoparticle-functionalized Janus micromotors, which are prepared by immobilizing uricase asymmetrically onto the surface of silicon particles, to actively and rapidly detect uric acid. The asymmetric distribution of uricase on silicon particles allows the Janus micromotors to display efficient motion in urine under the propulsion of biocatalytic decomposition of uric acid and simultaneously detect uric acid based on the luminescence quenching effect of the UCNPs modified on the other side of SiO 2 . The efficient motion of the motors greatly enhances the interaction between UCNPs and the quenching substrate and improves the uric acid detection efficiency. Overall, such a platform using uric acid simultaneously as the detected substrate and motion fuel offers considerable promise for developing multifunctional micro/nanomotors for a variety of bioassay and biomedical applications.

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL -1 ) -1  cm -2 , with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40 ng mL -1 ) at - 0.1 V (vs. Ag/AgCl), and a low detection limit of 2 pg mL -1 . The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H 2 O 2 , respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM -1  cm -2 ), and linear ranges (0.1-5, 0.1-6, 0.1-7 mM) with a detection limit of 10, 1, and 8 µM for glucose, uric acid, and H 2 O 2 , respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H 2 O 2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H 2 O 2 . Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H 2 O 2 ., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Nanozymes were the novel research field to replace natural enzymes because of stability and low cost. However, the research on nanozymes was mainly focused on peroxidase, and there was little research about nanozymes with oxidase-like activity, especially mimic oxidase of small molecules related to human physiology. High levels of uric acid (UA) in the body can cause hyperuricemia and gout. And natural uricase cured this disease because it could oxidize UA. The oxidase-like activity of mixed valence state metal organic frameworks with cerium (MVSM) had been studied, but MVSM was found to have uricase-like activity in this article. The catalytic process of UA with MVSM was studied by a variety of analytical methods, which was similar to the natural uricase except for further oxidation of H 2 O 2 . The catalytic activity constants of MVSM were acquired by the Michaelis-Menten equation. MVSM had a better ability to catalyze UA in in vivo and in vitro experiments. An enzyme-free analysis-based mimic uricase for UA was established. All the experimental results proved that MVSM had a good prospect to replace the natural uricase. A nanomaterial, mixed valence state Ce-MOF (MVSM), with uricase-like activity has been found in vivo and in vitro. This material has potential to be a fluorescent analysis for detecting uric acid without uricase., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Under physiological conditions, Bacillus fastidious uricase (BFU) activity shows negligible lagging phase before the exponential decrease; mutants are thus designed for long lagging phases before exponential activity decreases. On homodimer surface of BFU (4R8X.pdb), the last fragment ANSEYVAL at the C-terminus forms a loop whose Y319 is H-bonded by the buried D257 in the same monomer. Within 1.5 nm from the α-carboxyl group of the last leucine (L322), E30, K26, D257, R258, E311, K312 and E318 from the same monomer plus D126 and K127 from a monomer of the other homodimer generate an electrostatic interaction network. Within 1.5 nm from Y319, D307 and R310 in the same monomer interact with ionized residues around the inter-chain β-sheet in the same homodimer. Mutagenesis of Y319R is designed to strengthen the original interactions and concomitantly generate new electrostatic attractions between homodimers. Under physiological conditions, the mutant V144A/Y319R showed an approximately 4 week lagging phase before the exponential activity decrease, an apparent half-life of activity nearly three folds of mutant V144A, but comparable activity. The introduction of ionizable residues into the C-terminus contacting the other homodimer for additional and/or stronger electrostatic attractions between homodimers may be a universal approach to thermostable mutants of uricases., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microbial urate oxidase has emerged as a potential source of therapeutic properties for hyperuricemia in arthritic gout and renal disease. The thermostability and long-term thermal tolerance of the enzyme need to be established to prolong its therapeutic effects. Here, we present the biochemical and structural aspects of a hyperthermostable urate oxidase (TbUox) from the thermophilic microorganism Thermobispora bispora. Enzymatic characterization of TbUox revealed that it was active over a wide range of temperatures, from 30 to 70 °C, with optimal activity at 65 °C and pH 8.0, which suggests its applicability under physiological conditions. Moreover, TbUox exhibits high thermostability from 10 to 65 °C, with Tm of 70.3 °C and near-neutral pH stability from pH 7.0 to 8.0 and high thermal tolerance. The crystal structures of TbUox revealed a distinct feature of the C-terminal loop extensions that may help with protein stability via inter-subunit interactions. In addition, the high thermal tolerance of TbUox may be contributed by the extensive inter-subunit contacts via salt bridges, hydrogen bonds, and hydrophobic interactions. The findings in this study provide a molecular basis for the thermophilic TbUox urate oxidase for application in hyperuricemia and gout therapy., (Copyright © 2021. Published by Elsevier B.V.)

In the past two decades, the number of reports on the construction of uric acid (UA) sensors has increased dramatically, as it is a vital factor in the diagnosis of physiological functions and diseases. Among these sensors, cascade colorimetric detection based on peroxidase mimics has received great attention owing to the advantages of easier operation and more intuitive results. Herein, we report a simple UA detection method via the integration of urate oxidase (UOx) and PCN-222(Fe) with peroxidase-like activity, in which UOx is immobilized in PCN-222(Fe) by physical adsorption. UOx could catalyze the UA oxidation to produce H 2 O 2 , and then PCN-222(Fe) catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H 2 O 2 to generate a blue product with a maximum absorbance at 652 nm. Under optimal conditions, the increasing absorbance at 652 nm was proportional to the UA concentration. The linear range for UA detection was 10-800 μM with a limit of detection of 3.5 μM, and the cascade system has been successfully utilised for the detection of UA in human serum samples to demonstrate the accuracy of the proposed method. In conclusion, the cascade catalytic system based on the immobilization of UOx in PCN-222(Fe) has the potential to be used as a rapid and sensitive sensor for UA detection.

A simple dual-read assay for uric acid (UA) was developed based on a combined ratiometric fluorescent and colorimetric strategy using nitrogen-doped carbon dots (N-CDs). The biosensor relies on the oxidation of UA by uricase to produce H 2 O 2 , which was then converted to • OH radicals by I - , resulting in the oxidation of o-phenylenediamine (OPD) to 2,3-diaminophenazine (DAP). In the presence of UA, the colorless biosensor system changed to yellow. Furthermore, the presence of DAP quenched the fluorescence emission of the N-CDs at 427 nm based on the inner filter effect (IFE). With increasing UA concentrations, the fluorescence intensity of the biosensor at 427 nm decreased but increased at 580 nm, demonstrating the ratiometric response. A strong linearity was observed between the fluorescence intensity ratio of DAP to N-CDs (I 580 /I 427 ) and the corresponding UA concentration over the range 0.5-150 μM, and a limit of detection (S/N ratio of 3) of 0.06 μM was calculated. The dual-read assay was successfully employed in the quantitation of UA in human serum and urine samples, revealing its potential for measuring UA in clinical samples., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg 2+ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.

Urate oxidases (UOs) catalyze the cofactor-independent oxidation of uric acid, and an extensive water network in the active site has been suggested to play an essential role in the catalysis. For our present analysis of the structure and function of the water network, the crystal qualities of Bacillus sp. TB-90 urate oxidase were improved by controlled dehydration using the humid air and glue-coating method. After the dehydration, the P21212 crystals were transformed into the I222 space group, leading to an extension of the maximum resolution to 1.42 Å. The dehydration of the crystals revealed a significant change in the five-water-molecules' binding mode in the vicinity of the catalytic diad, indicating that these molecules are quasi-stable. The pH profile analysis of log(kcat) gave two pKa values: pKa1 at 6.07 ± 0.07 and pKa2 at 7.98 ± 0.13. The site-directed mutagenesis of K13, T73 and N276 involved in the formation of the active-site water network revealed that the activities of these mutant variants were significantly reduced. These structural and kinetic data suggest that the five quasi-stable water molecules play an essential role in the catalysis of the cofactor-independent urate oxidation by reducing the energy penalty for the substrate-binding or an on-off switching for the proton-relay rectification., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

A new ion-exchange medium was prepared from magnetic particles of ∼1.0 µm through coating with small zwitterions and then functionalizing with ampholytic groups for the isoelectric point of ∼6.4 and denoted MSP-ZEWB. With Meyerozyma guilliermondii uricase (MGU) as the model of soluble proteins, the purification of a protein via ion-exchange was compared between MSP-ZEWB through elution in discontinuous mode and Toyopearl SP-650C as a classical ion-exchange medium through elution in continuous mode. MGU was adsorbed at pH 7.6 or 8.0 and eluted via competitive displacement by NaCl or electrostatic repulsions with an elution buffer at pH 10 to reverse the type of net charges of MGU. From MSP-ZEWB, MGU was eluted more rapidly with the elution percentages higher than those from Toyopearl SP-650C. For yielding a unit of MGU activity, MSP-ZEWB gave the elution solution volumes that were ∼50% of those obtained with Toyopearl SP-650C. The yields of MGU of the highest purity from MSP-ZEWB were higher than those from Toyopearl SP-650C, but the highest purification folds with both media were comparable. MSP-ZEWB regenerated for 16 times still showed the consistent purification efficacy. Therefore, the ion-exchange media bearing small zwitterion coats showed great promise for the purification of soluble proteins.

Twenty-five microbial isolates were investigated for uricase production on uric acid medium. All isolates were obtained from Jeddah, Saudi Arabia. The highest uricase producer was identified as Bacillus cereus SKIII. Using glucose peptone broth at pH 7.5, incubation temperature 30 °C for 3 days with shaking of 150 rpm were the best conditions for maximum enzyme production. Glucose and peptone were the best carbon and nitrogen sources. The molecular weight of the purified enzyme was34.5 KDa, and isoelectric point was 7.9. The optimum pH and temperature were pH 8.0 and 35 °C, respectively. It was stable at 35 °C for 60 min, but thermally inactivated at 60 °C after 60 min Its enzymatic activity was enhanced by Mg 2+ , Ca 2+ ,Fe 2+ , Mn 2+, Zn 2+ ions and inhibited by Co 2+ , Na + , Hg 2+ , Ag + ions and EDTA at 1 mM. Uricase production was enhanced using UV mutation and the obtained mutant produced six times higher than the original isolate. An amplicon 900 bp of uricase gene (Pucl) was sequenced (accession number MF417635). No remarkable difference was noticed in B. cereus SKIII and SKm mutant nucleotide sequences. In conclusion, SKIII and SKm are promising strains in uricase production for biotechnological applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Electropolymerization of thionine (TH) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrodes (GCE) in ethaline deep eutectic solvent (DES) was carried out for the first time, to prepare poly(thionine) (PTH) films with different nanostructured morphologies. PTH films were formed on MWCNT/GCE by potential cycling electropolymerization in ethaline with the addition of different acid dopants CH 3 COOH, HClO 4 , HNO 3 , H 2 SO 4 and HCl, acetic acid being the best. The electropolymerization process was monitored with an electrochemical quartz crystal microbalance. The polymerization scan rate was a key factor affecting the electrochemical and morphological properties of the PTH Ethaline -CH 3 COOH/MWCNT/GCE; electrodeposition at 200 mV s -1 showing the best performance. The PTH/MWCNT/GCE platform was characterized using cyclic and differential pulse voltammetry, electrochemical impedance spectroscopy and scanning electron microscopy. The analytical characteristics of the PTH films were evaluated for sensing of ascorbic acid and biosensing of uric acid. The developed sensor exhibited a low detection limit (1.1 μM), wide linear range (2.8-3010 μM) and high sensitivity (1134 μA cm -2  mM -1 ) for ascorbic acid. After immobilization of uricase, UOx, on PTH/MWCNT/GCE, the biosensor was successfully applied to the determination of uric acid, with fast response (˂ 7 s), good sensitivity (450 μA cm -2  mM -1 , wide linear range (0.48-279 μM) and low detection limit (58.9 nM), better than in the literature and than with PTH prepared in aqueous solution. The determination of uric acid in synthetic urine samples was successfully tested and the mean analytical recovery was 100.8 ± 1.4%. This is a promising approach for the determination of uric acid in real samples. Graphical abstract.

The amounts of uric acid (UA) in non-invasive biological samples, such as saliva, are critical for diagnosis and therapy of gout, hyperuricemia, Lesch-Nyhan syndrome, and several other diseases. Here, disposable UA biosensors were fabricated with the screen printing technique on the substrate of flexible PET. The working electrode was modified with carbon nanotubes followed by uricase for UA detection with excellent selectivity. The biosensor showed good electrocatalytic activity toward UA with high sensitivity, low detection limit, and wide linear range, which covers the full range of UA levels in human saliva. We demonstrate that UA can be directly detected in human saliva with the biosensor and the experimental data were consistent with the clinical analysis. This study indicated that the non-invasive biosensor is an attractive and possible approach for the monitoring of salivary UA. Graphical abstract A disposable uric acid biosensor modified with carbon nanotubes followed by uricase was fabricated on flexible PET and applied for the monitoring of salivary uric acid in human saliva.

Objective: The relationship between serum uric acid (SUA) and the risk of ischemic stroke (IS) has been fully elucidated in previous studies. Therefore, we further investigated the relationship between SUA levels and the risk of IS., Patients and Methods: 2195 patients at the Beijing Hospital, between February 2012 and May 2018, were enrolled in our hospital-based cross-sectional study. The patients were divided into an IS group and a (non-IS) control group, based on their medical records. SUA level was measured using the enzymatic uricase method. Univariate and multivariable logistic regression models were used for the analysis., Results: A total of 300 patients with IS [176 men; age (mean ± SD): 71.38 ± 10.66 years] and 1895 control patients [1060 men; age (mean ± SD): 66.12 ± 12.04 years] were enrolled in this study. IS patients had higher concentrations of SUA, compared with control group patients [6.11 ± 1.92 vs. 5.77 ± 1.62 (mg/dL)]; P = 0.004). We observed a J-shaped association between SUA levels and the risk of IS. Both the univariate and multivariate logistic regression analyses found a significantly elevated risk of IS in the bottom and upper SUA levels both in quartiles and deciles, compared with the intermediate SUA levels., Conclusion: These results indicate a J-shaped, independent association between SUA levels and the risk of IS in high-risk individuals., (Copyright © 2020 Elsevier B.V. All rights reserved.)