Urate oxidase (Uox) plays a pivotal role in uric acid (UA) degradation, and it has been applied in controlling serum UA level in clinical treatment of hyperuricemia (HUA). However, because Uox is a heterogenous protein to the human body, the immune rejections typically occur after intravenous administration, which greatly hampers the application of Uox-based agents. In this study, we used Lactococcus lactis NZ9000, a food-grade bacterium, as a host to express exogenous Uox genes, to generate the Uox-expressing engineered strains to treat HUA. Aspergillus flavus-derived Uox (aUox) and the "resurrected" human-derived Uox (hUox) were cloned into vector and expressed in NZ9000, to generate engineered strains, respectively. The engineered NZ9000 strains were confirmed to express Uox and showed UA-lowering activity in a time-dependent manner in vitro. Next, in an HUA mice model established by oral gavage of yeast paste, the UA levels were increased by 85.4% and 106.2% at day 7 and day 14. By contrast, in mice fed with NZ9000-aUox, the UA levels were increased by 39.5% and 48.3% while in mice fed with NZ9000-hUox were increased by 57.0% and 82.9%, suggesting a UA-lowering activity of both engineered strains. Furthermore, compared with allopurinol, the first-line agent for HUA treatment, mice fed with NZ9000-aUox exhibited comparable liver safety but better kidney safety than allopurinol, indicating that the use of engineered NZ9000 strains not only alleviated kidney injury caused by HUA, but could also avoided the risk of kidney injury elicited by using allopurinol. Collectively, our study offers an effective and safe therapeutic approach for HUA long-term treatment and controlling.
Animals ; Lactococcus lactis/metabolism* ; Urate Oxidase/genetics* ; Mice ; Uric Acid/blood* ; Hyperuricemia ; Humans ; Administration, Oral ; Aspergillus flavus/genetics* ; Male

OBJECTIVETo characterize the property of uricase loaded in uricase-catalase liposomes (BUCLPs) prepared using borate buffer.

METHODSBUCLPs were prepared using reverse-phase evaporation, and the physicochemical properties of uricase in the prepared BUCLPs were examined.

RESULTSThe optimal temperature of BUCLP and URI was 40 degrees celsius, their optimal pH values were 8.0 and 8.5, and their Michaelis-Menten constants were 14.207 µmol/L and 13.623 µmol/L, respectively. Fluorescence intensity of nanoliposome-loaded uricase-catalase that bound to FITC was higher than that of uricase-catalase binding directly with FITC; the fluorescence intensity of BUCLP was higher than that of free uricase-catalase at 280 nm.

CONCLUSIONUricase activity is enhanced after loading in uricase and catalase liposomes.

Borates ; Catalase ; Liposomes ; Nanoparticles ; chemistry ; Temperature ; Urate Oxidase ; chemistry

Aims: The present study aimed at isolating new source of uricase producers from Malaysian hot springs together with partial purification and characterization of thermophilic uricase from novel strain. Methodology and results: A bacteria strain, designated as SN4, was found to have the ability to degrade uric acid. 16S rRNA analysis identified SN4 as Pseudomonas otitidis. Uricase was then extracted from SN4 and purification was performed via ammonium sulphate precipitation. The effects of temperature, pH and metal ions on partially purified uricase were evaluated. Results showed that 70% ammonium sulphate concentration gave the highest uricase activity at 4.18 U/mL compared to other concentrations. The molecular weight of the partially purified uricase was 33 kilodalton (kDa). The optimum temperature for uricase was 45 °C and its activity was highest at pH 8.0. Calcium ions and copper ions enhanced uricase activity while cobalt ions reduced uricase activity. Conclusion, significance and impact of study: Isolation and investigation of uricase producers from new sources such as thermophiles would increase availability and thermal stability of the uricase that could be used for significant purposes such as in biochemical and clinical applications.
Urate Oxidase

In order to characterize a thermostable urate oxidase (Uox) from Microbacterium sp. strain ZZJ4-1, we cloned its gene (uox). The open reading frame of uox contained 894 base pairs and encoded a protein with 297 amino acids. Alignment of gene sequences indicated there was no obvious identity with the most reported uox and that 72% identity was found with uox from Arthrobacter globiformis. We inserted the gene into the plasmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 (DE3). After the purification of the recombinant Uox by the HisBind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. It was stable below 65 degrees C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag+, Zn2+, CU2+ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.
Actinomycetales ; enzymology ; genetics ; Amino Acid Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Urate Oxidase ; genetics ; metabolism

There is an increasing incidence of gout and hyperuricemia worldwide. It is because the population is getting older, their life style is sedentary, and they take protein-enriched food. Gout is one of the most common but best controllable chronic diseases of adult. There have been recent advances in the understanding of underlying mechanisms and treatment of gout and hyperuricemia. This article is aimed to provide the practical review of the currently recommended practice of care and also to introduce some recently approved drugs. The management concept of hyperuricemia is changing because not only the gout but also the hyperuricemia appear to be independent risk factors for hypertension, renal disease and cardiovascular disease. Gout causes a significant individual and social burden and loss of working force. Still hyperuricemia in the gout patient is often under-treated by the patients themselves and by the physicians also. Once the acute gout attack is controlled, patients should be followed with goal-oriented treatment of hyperuricemia and other risk factors. Allopurinol has remained as a first-line treatment for chronic hyperuricemia, but uricosuric agents may also be considered in some patients. These drugs have provided good control of the disease in most gout patients until now but the elderly patients with gout often carry co-medications, contra-indication to these drugs, and risk of adverse drug reaction. Febuxostat is a nonpurine xanthine-oxidase inhibitor. It is a new agent approved by the US FDA and Korean FDA for the treatment of hyperuricemia in patients with gout which may be used when allopurinol is not tolerated or contraindicated. Pegloticase is the PEGylated urate oxidase which is very potent and so recently approved by the US FDA for the gout refractory to conventional treatment.
Adult ; Aged ; Allopurinol ; Arthritis, Gouty ; Cardiovascular Diseases ; Chronic Disease ; Drug Toxicity ; Gout ; Humans ; Hypertension, Renal ; Hyperuricemia ; Incidence ; Life Style ; Polyethylene Glycols ; Risk Factors ; Thiazoles ; Urate Oxidase ; Uricosuric Agents ; Febuxostat

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.
Aspergillus flavus ; enzymology ; Cloning, Molecular ; Codon ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification ; Uric Acid ; metabolism

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.
Animals ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Swine ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification

Gout is a common chronic inflammatory arthritis that can lead to significant disability. Gout is one of the few rheumatologic diseases that can be diagnosed with certainty and can be cured with appropriate therapy. Alcohol and dietary consumption are related to hyperuricemia and gout attacks. A moderate intake of purine-rich vegetables or protein is not related to an increased risk of gout. A weight-reducing, calorie-restricted diet with moderate carbohydrate restriction was beneficial for gout patients and reduced the serum uric acid and frequency of gout attacks, although these findings need to be confirmed. Clinicians should consider therapeutic options that do not increase the serum uric acid when treating associated conditions in gout patients. The acute gout attack can be treated appropriately with non-steroidal anti-inflammatory drugs, colchicine, or glucocorticoids. Hypouricemic treatment reduces the uric acid concentration by inhibiting its production (allopurinol) or enhancing its excretion (benzbromarone). Allopurinol is the agent used most commonly, but the recommended dose often fails to control the serum uric acid. Benzbromarone effectively reduces the serum uric acid, but possible hepatotoxicity should be monitored. Febuxostat, a new xanthine oxidase inhibitor, was recently approved by the Federal Drug Administration (FDA). PEGylated uricase, a potent parenteral hypouricemic agent, is under investigation for the treatment of gout.
Allopurinol ; Arthritis ; Benzbromarone ; Colchicine ; Diet ; Glucocorticoids ; Gout ; Humans ; Hyperuricemia ; Thiazoles ; Urate Oxidase ; Uric Acid ; Vegetables ; Xanthine Oxidase ; Febuxostat

INTRODUCTIONHyperuricaemia in tumour lysis syndrome (TLS) can cause acute renal failure (ARF), necessitating dialysis. Recombinant urate oxidase (rasburicase) converts uric acid to soluble allantoin, which is excreted easily.

CASE REPORTAn 8-year-old boy with stage 3 Burkitt's lymphoma, TLS was successfully treated with hyper-hydration, diuretics and rasburicase, without dialysis. This is the first paediatric case in Kandang Kerbau Women's & Children's Hospital (KKH) in which rasburicase was used. We review the literature on the effectiveness of urate oxidase in avoiding dialysis in TLS.

TREATMENT AND OUTCOMEOur patient developed rapidly rising serum uric acid (SUA) and progressive renal impairment. Hyper-hydration and rasburicase (0.2mg/kg) were administered. SUA rapidly decreased from 1308 to 437 mmol/L within 12 hours. Urate oxidase has shown better results than allopurinol. There was a need for dialysis in 0.4% to 1.7% of patients with haematological malignancies given rasburicase, compared to 20% in patients given allopurinol.

CONCLUSIONSRasburicase can reverse renal insufficiency. Though expensive, it may be cost-effective by lowering incidence of dialysis, shortening the duration of intensive care and hospitalisation, allowing early chemotherapy.

Burkitt Lymphoma ; complications ; Child ; Humans ; Hyperuricemia ; drug therapy ; Male ; Renal Dialysis ; Singapore ; Treatment Outcome ; Tumor Lysis Syndrome ; physiopathology ; urine ; Urate Oxidase ; metabolism ; pharmacology ; Uric Acid ; analysis ; blood

A patented kinetic uricase method was evaluated for serum uric acid assay. Initial absorbance of the reaction mixture before uricase action (A(0)) was obtained by correcting the absorbance at 293 nm measured before the addition of uricase solution, and background absorbance (A(b)) was predicted by an integrated method. Uric acid concentration in reaction solution was calculated from A, the difference between A(0) and A(b), using the absorptivity preset for uric acid. This kinetic uricase method exhibited CV<4.3% and recovery of 100%. Lipids, bilirubin, hemoglobin, ascorbic acid, reduced glutathione and xanthine <0.32 mmol/L in serum had no significant effects. A linearly responded to 1.2 to 37.5 micromol/L uric acid in reaction solution containing 15 microl serum. The slope of linear response was consistent with the absorptivity preset for uric acid while the intercept was consistent with that for serum alone. Uric acid concentrations in clinic sera by different uricase methods positively correlated to each other. By Bland-Altman analysis, this kinetic uricase method accorded with that by quantifying the total change of UV absorbance on the completion of uricase reaction. These results demonstrated that this kinetic uricase method is reliable for serum uric acid assay with enhanced resistance to both xanthine and other common errors, wider range of linear response and much lower cost.
Humans ; Kinetics ; Reagent Kits, Diagnostic ; Spectrophotometry, Ultraviolet ; Urate Oxidase ; chemistry ; Uric Acid ; blood ; metabolism