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Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, also in prophylaxis and treatment of tumor lysis and organ transplants hyperuricemia. Uricase is commonly used as diagnostic reagent in clinical analysis for quantification of uric acid in blood and other biological fluids. Also, it can be used as an additive in formulations of hair coloring agents. A newly isolated strain, Aspergillus sp. 1-4, was able to produce extracellular uricase on a medium containing uric acid as inducer. Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1-4 is closely related to Aspergillus welwitschiae and its nucleotide sequence was deposited in the GenBank database and assigned sequence accession number MG323529. Statistical screening using Plackett-Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae. Results of statistical analysis indicated that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.48, 5.26 and 4.75; respectively. The interaction effects and optimal levels of these factors were evaluated using central composite design. The maximum uricase production was achieved at incubation time (5 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) are the optimum levels for maximum uricase production (60.03 U/mL). After optimization, uricase production increased by 3.02-folds as compared with that obtained from the unoptimized medium (19.87 U/mL).

Urate oxidase (Uox), an enzyme catalyzing oxidation of uric acid to allantoin, is widely used as diagnostic reagents and for treatments of uarthritis and hyperuricemia diseases. In our study, a higher Uox producer, bacterial strain OUC-1, was isolated from soil samples. The 16S rRNA gene sequence of strain OUC-1 showed 99% identity to the homologous fragments of Bacillus fastidiosus. After purification, Uox showed the optimal pH and temperature was 10.0 and 40 °C. The Km value of Uox was (0.15±0.04) mmol/L (n=5) with uric acid as the substrate. Uox activity was enhanced by Mg²⁺, and seriously inhibited by Zn²⁺ and SDS. Then the uox gene of B. fastidiosus OUC-1 was amplified and sequenced. The 3D structures of Uox, predicted with SWISS-MODEL, showed a homotetramer structure with a subunit molecular weight of 35.38 kDa. Finally, the gene coding for the B. fastidiosus Uox was successfully cloned and heterologously expressed in E. coli, which provides theoretical basis and technical support for improvement of Uox in the future.

Low energy ultrasound irradiation was used to enhance co-production of enzymes uricase and alkaline protease using Bacillus licheniformis NRRL 14209. Production of uricase and alkaline protease was evaluated for different ultrasound parameters such as ultrasound power, time of irradiation, duty cycle and growth stage of organisms at which irradiation is carried out. Maximum uricase production of 0.825 U/mL and alkaline protease of 0.646 U/mL have been obtained when fermentation broth was irradiated at 6 h of growth stage with 60 W power for 15 min of duration having 40% of duty cycle. The enzyme yield was found to be enhanced by a factor of 1.9-3.8 and 1.2-2.2 for uricase and alkaline protease respectively. Nevertheless, intracellular uricase was also observed in a fermentation broth after ultrasonic process intensification. The results indicate the effectiveness of low frequency ultrasound in improving enzyme yields with a vision of commercial applicability of the process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Uricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency of Bacillus cereus to produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41 U/ml uricase in medium containing 2.0 g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150 rpm in 26 h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58 U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1 mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K +  > Ca 2+  > Zn 2+  > Fe 3+  > Ni 2+  > Mg 2+  > Mn 2+  > Cu 2+ .

The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C.

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques., (© 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)

Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

Background: Intestinal microbiome constitutes a symbiotic ecosystem that is essential for health, and changes in its composition/function cause various illnesses. Biochemical milieu shapes the structure and function of the microbiome. Recently, we found marked differences in the abundance of numerous bacterial taxa between ESRD and healthy individuals. Influx of urea and uric acid and dietary restriction of fruits and vegetables to prevent hyperkalemia alter ESRD patients' intestinal milieu. We hypothesized that relative abundances of bacteria possessing urease, uricase, and p-cresol- and indole-producing enzymes is increased, while abundance of bacteria containing enzymes converting dietary fiber to short-chain fatty acids (SCFA) is reduced in ESRD., Methods: Reference sets of bacteria containing genes of interest were compiled to family, and sets of intestinal bacterial families showing differential abundances between 12 healthy and 24 ESRD individuals enrolled in our original study were compiled. Overlap between sets was assessed using hypergeometric distribution tests., Results: Among 19 microbial families that were dominant in ESRD patients, 12 possessed urease, 5 possessed uricase, and 4 possessed indole and p-cresol-forming enzymes. Among 4 microbial families that were diminished in ESRD patients, 2 possessed butyrate-forming enzymes. Probabilities of these overlapping distributions were <0.05., Conclusions: ESRD patients exhibited significant expansion of bacterial families possessing urease, uricase, and indole and p-cresol forming enzymes, and contraction of families possessing butyrate-forming enzymes. Given the deleterious effects of indoxyl sulfate, p-cresol sulfate, and urea-derived ammonia, and beneficial actions of SCFA, these changes in intestinal microbial metabolism contribute to uremic toxicity and inflammation.

Objective: To construct a plasmid containing a urate oxidase and creatinine hydrolase fusion gene and transform the plasmid into Escherichia coli to decompose uric acid and creatinine., Methods: According to the GenBank data for the urate oxidase gene, specific primers were designed to amplify and remove the stop codon for the urate oxidase gene. The gene was then ligated into the plasmid pMG36e to construct pMG36e-U. Then, using the GenBank database for the creatinine hydrolase gene, primers were designed to amplify the creatinine hydrolase gene. This gene was ligated into pMG36e-U to form pMG36e-U/C. Next, this construct was transformed into E. coli, which was confirmed by screening the recombinant E. coli and sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The engineered bacteria were cultured with a specific concentration of creatinine and uric acid for 24 h. Then, the concentrations of creatinine and uric acid in the culture fluid were measured., Results: The recombinant gene fragment was approximately 1.68 kb, and it contained the urate oxidase and creatinine hydrolase genes. The transformed E. coli expressed creatinine hydrolase and uric acid oxidase. The creatinine decomposition rate increased by 43.5%, and the uric acid decomposition rate increased by 42.32%., Conclusion: The constructed recombinant plasmid containing a fusion gene of creatinine hydrolase and uric acid oxidase was transformed into E. coli, and the enzymatic activities were expressed.

Background: The detection and quantification of uric acid in human physiological fluids is of great importance in the diagnosis and therapy of patients suffering from a range of disorders associated with altered purine metabolism, most notably gout and hyperuricaemia. The fabrication of cheap and reliable urate-selective amperometric biosensors is a challenging task., Results: A urate-selective microbial biosensor was developed using cells of the recombinant thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element. The construction of uricase (UOX) producing yeast by over-expression of the uricase gene of H. polymorpha is described. Following a preliminary screening of the transformants with increased UOX activity in permeabilized yeast cells the optimal cultivation conditions for maximal UOX yield namely a 40-fold increase in UOX activity were determined.The UOX producing cells were coupled to horseradish peroxidase and immobilized on graphite electrodes by physical entrapment behind a dialysis membrane. A high urate selectivity with a detection limit of about 8 μM was found., Conclusion: A strain of H. polymorpha overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed.

The strain PNR11 was isolated from gut of termite during the screening for uric acid degrading actinomyces. This strain was able to produce an intracellular uricase when cultured in fermentation medium containing uric acid as nitrogen source. Base on its morphological characters and 16S rDNA sequence analysis, this strain belong to the genus Saccharopolyspora. This is the first report ofuricase produced from the genus Saccharopolyspora. The aim of this study was to investigate the effects of different factors on uricase production by new source of Saccharopolyspora. Saccharopolyspora sp. PNR11 was cultured in production medium in order to determine the best cultivation period. The result showed that the time period required for maximum enzyme production was 24 h on a rotary shaker operating at 180 rpm. Optimized composition of the production medium consisted of 1% yeast extract, 1% maltose, 0.1% K2HPO4, 0.05% MgSO4 7H2O, 0.05% NaCl and 1% uric acid. The optimum pH and temperature for uricase production in the optimized medium were pH 7.0 and 30 degrees C, respectively. When the strain was cultured at optimized condition, the uricase activity reached to 216 mU mL(-1) in confidential level of 95%. The crude enzyme had an optimum temperature of uricase was 37 degrees C and it was stable up to 30 degrees C at pH 8.5. The optimum pH ofuricase was 8.5 and was stable in range of pH 7.0-10.0 at 4 degrees C. This strain might be considered as a candidate source for uricase production in the further studies. Present finding could be fulfill the information ofuricase produce from actinomycetes.

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.

Uric acid (UA) results from xanthine oxidase (XO) catabolism of xanthine and is the final product of purine catabolism in humans. In this species, hyperuricemia is associated with gout, nephropathy, and increased cardiovascular disease risk. Although the effects of hyperuricemia in vascular biology are overall controversial, UA has been described as an antioxidant and as potentially improving endothelial function. Hypertension is associated with endothelial dysfunction. We hypothesized that UA improves the endothelial function of aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. UA (100 microM) in the presence of the uricase inhibitor oxonic acid (10 microM) did not modify relaxation to acetylcholine (ACh) (1 nM-10 microM) in the aorta from nontreated, sham normotensive, and DOCA-salt hypertensive rats [response to 10 microM ACh for UA versus vehicle, respectively: nontreated = 37 +/- 7 versus 48 +/- 7%, sham = 53 +/- 15 versus 57 +/- 20%, DOCA = 81 +/- 4 versus 85 +/- 2% from 20 microM prostaglandin 2alpha (PGF(2alpha))-induced contraction]. Allopurinol (100 microM), a XO inhibitor, did not significantly alter the ACh-induced relaxation of sham and DOCA aortic rings (response to 10 microM ACh for allopurinol versus vehicle, respectively: sham = 61 +/- 5 versus 68 +/- 9%, DOCA = 87 +/- 6 versus 88 +/- 3% from 20 microM PGF(2alpha)-induced contraction). Uricemia, ranging from unmeasurable to 547 microM in sham and to 506 microM in DOCA rats, was not significantly different between these two groups. The expression and activity of XO, as well as the expression of uricase, were not different between sham and DOCA rat aorta. We conclude that, at least in vitro, UA does not affect the ACh-induced relaxation of normotensive and DOCA-salt hypertensive rats.

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.

The peroxisomal enzyme urate oxidase plays a pivotal role in the degradation of purines in both prokaryotes and eukaryotes. However, knowledge about the purine-induced expression of the encoding gene is lacking in vertebrates. These are the first published sequences of fish urate oxidase, which were predicted from PCR amplified liver cDNAs of Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus) and African lungfish (Protopterus annectens). Sequence alignment of different vertebrate urate oxidases revealed amino acid substitutions of putative functional importance in the enzyme of chicken and lungfish. In the adult salmon, expression of urate oxidase mRNA predominated in liver, but was also identified in several nonhepatic organs including brain, but not in skeletal muscle and kidney. Juvenile salmon fed diets containing bacterial protein meal (BPM) rich in nucleic acids showed a significant increase in liver urate oxidase enzyme activity, and urea concentrations in plasma, muscle and liver were elevated. Whereas salmon fed the 18% BPM diet showed a nonsignificant increase in liver mRNA levels of urate oxidase compared with the 0% BPM-fed fish, no further increase in mRNA levels was found in fish receiving 36% BPM. The discrepancy between urate oxidase mRNA and enzyme activity was explained by rapid mRNA degradation or alternatively, post-translational control of the activity. Although variable plasma and liver levels of urate were detected, the substrate increased only slightly in 36% BPM-fed fish, indicating that the uricolytic pathway of Atlantic salmon is intimately regulated to handle high dietary purine levels.

In tropical legumes like Glycine, Phaseolus and Vigna sp., ammonia as direct product of symbiotic nitrogen fixation is converted to ureides (allantoin and allantoic acid) and they were translocated to the shoots as nitrogen source. In the xylem sap of soybean in reproductive phase the ureides reached to 60-75% of soluble nitrogen. In nodules infected cells (plastid and mitochondria) and uninfected cells (peroxisome) shares de novo purine biosynthesis and urate oxidation to produce ureides respectively. Current research revealed unique feathers on this symbiotic metabolism, especially on regulation of purine biosynthesis, uricase gene expression and feedback inhibition of ureides to nitrogen fixing activity.

Long-term treatment of rodents with peroxisome proliferator chemicals, a group of structurally diverse nongenotoxic carcinogens, leads to liver cancer in a process dependent on the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha). Previous in vitro studies have shown that growth hormone (GH) can inhibit PPARalpha-dependent gene expression by down-regulation of PPARalpha expression and by a novel inhibitory cross-talk involving the GH-activated transcription factor STAT5b. Presently, we evaluate the role of STAT5b in mediating these inhibitory actions of GH on PPAR function using a STATb-deficient mouse model. Protein levels of three PPARalpha-responsive peroxisomal beta-oxidation pathway enzymes (fatty acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and L-bifunctional enzyme) were increased up to two- to threefold in STAT5b(-/-) relative to wild-type control mouse liver, as was the basal expression of two PPARalpha-regulated cytochrome P450 4A proteins. In contrast, protein levels of two PPARalpha-unresponsive peroxisomal enzymes, catalase and urate oxidase, were not affected by the loss of STAT5b. A corresponding increase in expression of fatty acyl-CoA oxidase and L-bifunctional enzyme mRNA, as well as PPARalpha mRNA, was observed in the STAT5b-deficient mice, suggesting a transcriptional mechanism for the observed increases. Although basal liver expression of PPARalpha and its target genes was thus elevated in STAT5b(-/-) mice, the clofibrate-induced level of enzyme expression was unaffected, suggesting that the inhibitory effects of STAT5b are overcome at high concentrations of PPARalpha activators. These findings support the hypothesis that GH and potentially other endogenous activators of STAT5b help to maintain liver PPARalpha function at a low basal level and may thereby moderate PPARalpha-dependent hepatocarcinogenesis and other responses stimulated by exposure to low levels of environmental chemicals of the peroxisome proliferator class.

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus, A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, although no significantly increased uricase production was found.

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus. A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for both A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.

Anurate oxidase (uricase, EC 1.7.3.3) gene from Candida utilis AS2.117 was cloned by PCR amplification with primers derived from conserved regions of published uicase DNA sequence. The DNA sequence of cloned uricase gene was determined and a high homology compared to the reported gene was found. The cloned gene was inserted into Bam H I and Nde I sites of pET21a to create the recombinant plasmid pURO. In Escherichia coli BL21(DE3) host, the expression lever of uricase reached to about 40% of total soluble proteins of the cell. The western blot analysis confirmed the result of expression. Properties of the enzyme protein produced by E. coli BL21(DE3)/pURO were determined and similar with those of original protein from Candida utilis AS2.117. Furthermore, the thermostability of the expressed protein was enhanced. The purified recombinant uricase was used in serum uric acid analysis.

Wild-type and mutated urate oxidase (UO) proteins were overexpressed in Cos-1 and HEK293 cells and were analyzed by Western blotting and several morphological methods. By immunoelectron microscopy, wild-type UO formed large aggregates in the cytoplasm and nucleoplasm and exhibited a crystalloid structure. Mutated UO (UOdC), from which 28 amino acids, including peroxisomal targeting signal at the C-terminus, were deleted, formed dispersed aggregates in the cytoplasm and nucleus. Chimeric UO (MUOdC), which was made by addition of the mitochondrial targeting signal of serine:pyruvate/glyoxylate aminotransferase to the N-terminus of UOdC, attached to ER to form a complicated MUOdC-ER complex. These three structures were immunostained for ubiquitin- and p32-subunits of proteasomes. Western blotting showed strong signal for UO and UOdC but very weak signal for MUOdC. The results suggest that overexpressed UO and UOdC accumulate in the cells because their synthesis rate is higher than the degradation rate, whereas MUOdC forming a complex with ER is degraded very rapidly. The ubiquitin-proteasome pathway may be involved in the degradation of these proteins.

Uricase II is a purine metabolic enzyme highly induced in root nodules during the symbiosis established between legumes and bacteria of the genera Rhizobium and Bradyrhizobium. Here we describe the characterization of bean (Phaseolus vulgaris) nodule uricase II cDNA and show that uricase II is encoded by a single gene in the bean genome. This gene is also expressed in cotyledons, roots, and hypocotyls during bean seedling establishment, and an anti-uricase antibody recognizes the protein in different seedling organs. Uricase II has also been found in Leucaena leucocephala seedlings, suggesting that it participates during seedling establishment in legumes that do not transport ureides. A 50-kD polypeptide that is detected by the anti-uricase antibody is found in cotyledons during seedling development. This higher-molecular-mass form is also detected in developing roots and hypocotyls but not in nodules. In situ hybridization experiments in root seedlings showed uricase II transcripts in the metaxylem parenchyma cells and phloem fibers of the vascular system.

To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strains. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

Uricase from Cellulomonas flavigena SK-4 is an industrially useful enzyme for commercial formulations of hair coloring. The uricase production by recombinant Escherichia coli strain with a high cell density cultivation technique was described. Of three kinds of media, synthetic media with the feeding of a high concentration of glucose solution were suitable for high cell density cultivation. As for feeding, both biomass concentration and uricase productivity were increased by about two (61.2 g dry cell weight (DCW)/liter) and three times (1037 U/ml broth), respectively, in 24 h by continuous supply. In the case of feeding by a DO-stat method, however, cell concentration was comparable to continuous glucose supply but uricase activity was reduced. By supplying pure oxygen to compensate for oxygen limitation during cultivation, the highest values of 77.4g DCW/liter and 1113 U/ml broth of the uricase activity were achieved with the total cultivation time of 15 h.

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26 degrees C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C-terminal 10 amino acid residues of acyl-CoA oxidase (the C-terminal tripeptide is Ser-Lys-Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C-terminal Ser-Lys-Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C-terminal Ser-Arg-Leu (SRL) sequence was deleted or mutated to Ser-Glu-Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C-terminus the import activity was retained. These results show that urate oxidase is important into peroxisomes via a common pathway with acyl-CoA oxidase, and that the C-terminal SRL sequence functions as a peroxisomal-targeting signal.

Urate oxidase, an enzyme used in human therapy, is currently produced industrially by a strain of Aspergillus flavus. Two strategies of strain improvement were tested in order to obtain higher yields of urate oxidase. The first one, based on a classical mutation-selection protocol, led to the isolation of a mutant strain that overproduced uricase two-fold as compared to the industrial strain. The second one consisted in the construction of transformed strains that had integrated multiple copies of a urate oxidase-expression vector. A twenty-fold improvement in urate oxidase was obtained by this method.

Strains of Saccharomyces cerevisiae producing Aspergillus flavus uricase (Uox) have been constructed. An artificial promoter which combined the upstream and downstream sequences of the GAL7 and ADH2 promoters, respectively, was found to be efficient in directing the synthesis of uaZ mRNAs encoding Uox. A good proportionality between the copy number of the uaZ expression cassette and the level of Uox production was found in the range of 1-10 copies. Transformants accumulated active and soluble Uox to a level exceeding 13% of total protein, as deduced from enzymatic assays. This relative level could be improved two- to threefold by using a recipient strain in which the wild-type GAL4 gene had been deleted and which expressed a GAL4 construct placed under the control of the ADH2 promoter.

Urate oxidase, an enzyme involved in purine catabolism, comprises the crystalline core of rat liver peroxisomes. An affinity-purified monospecific antibody was developed to study the expression of urate oxidase protein levels. Immunoreactive urate oxidase was not detectable in prenatal liver; however, it is present at low levels after birth until approximately day 15 (postnatal age); expression sharply increases just prior to day 20, after which the enzyme is maintained at adult levels. This pattern of expression was similar to that of another peroxisomal enzyme, catalase; these developmental increases reflect the increase in peroxisomal number. Administration of exogenous glucocorticoid hormone to 10-day-old rats resulted in a precocious rise (2.5-fold) in urate oxidase levels. Adrenalectomy at 10 days of age did not cause decreased levels in the fourth week of life. In adult animals, while exogenous glucocorticoid administration did not influence urate oxidase levels, adrenalectomy at 60 days of age decreased urate oxidase levels to 40 percent of control levels. Subsequent administration of exogenous glucocorticoid hormone restored urate oxidase to normal levels. Parallel studies of catalase levels indicate that this glucocorticoid-sensitive response is not generalized for all peroxisomal proteins. Our results suggest that peroxisomes proliferate during early postnatal development, but after this process is complete, the biogenesis of individual peroxisomal proteins may be independently regulated.

The effect of lowering oxygen concentration on the expression of nodulin genes in soybean callus tissue devoid of the microsymbiont has been examined. Poly(A)+ RNA was isolated from tissue cultivated in 4% oxygen and in normal atmosphere. Quantitative mRNA hybridization experiments using nodule-specific uricase (Nodulin-35) and sucrose synthase (Nodulin-100) cDNA probes confirmed that the synthesis of the uricase and sucrose synthase is controlled by oxygen at the mRNA level. The steady-state levels of uricase and sucrose synthase mRNA increased significantly (5-6- and 4-fold respectively) when the callus tissue was incubated at reduced oxygen concentration. Concomitant with the increase in mRNA level a 6-fold increase in specific activity of sucrose synthase was observed. Two messengers representing poly-ubiquitin precursors also responded to lowering the oxygen concentration. The increase was about 5-fold at 4% oxygen. No expression at atmospheric oxygen or in response to low oxygen was observed when using cDNA probes for other nodulin genes such as leghemoglobin c3, nodulin-22 and nodulin-44.

Male Wistar rats were given 200 mg/kg/day nicotinic acid or 1000 mg/kg/day cholestyramine by stomach tube for ten days. Peroxisomal palmitoyl-CoA oxidation (cyanide-insensitive) and the activities of palmitoyl-CoA hydrolase and urate oxidase were significantly increased in the total liver homogenate. Subcellular fractionation showed enhanced enzyme activities after drug treatment mainly in the peroxisome-containing fractions. The increase in urate oxidase activity and its subcellular distribution suggest that the tested drugs induce core-containing peroxisomes. The findings are similar to those previously reported with low doses of peroxisome-proliferating hypolipidemic drugs and with acetylsalicylic acid, a drug which is structurally similar to nicotinic acid. Since cholestyramine is not absorbed, its influence on hepatic enzymes probably occurs indirectly as a consequence of enhanced catabolism of cholesterol.

The processes associated with the biogenesis of peroxisomes in mouse liver have been studied by following the incorporation of radiolabelled leucine into major enzymic components of this organelle. Maximal incorporation of label into peroxisomal catalase and urate oxidase occurred within 2 hr, with the urate oxidase being labelled before catalase, but subsequent to the incorporation of phospholipid into this organelle. Subsequently, immunoprecipitation of catalase from the large granular fraction of mouse liver was shown to result in the isolation of a catalase molecule which had lost a peptide of approx. 2000 dalton from each subunit by comparison with the newly-synthesized enzyme. It was observed that the modification of catalase was obviated by the presence of leupeptin and iodoacetamide and this information has enabled the purification of both modified and unmodified forms of the enzyme. The possible significance of these data has been discussed and the major features incorporated into a working model of peroxisomal biogenesis.

Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.

Five mutants were isolated at the all2 gene on the basis of their inability to utilize hypoxanthine as a sole source of nitrogen. These mutants failed to utilize the purines adenine, hypoxanthine, xanthine, uric acid, allantoin and allantoic acid, although they could utilize urea and ammonium. The all2 mutants appeared to be defective in purine induction of uricase, allantoinase, allantoicase and ureidoglycollase activities but retained wild-type activity of the constitutively synthesized urease. The all2 mutations were recessive.

The intracellular site of synthesis of two peroxisomal enzymes of rat liver, uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) and catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6), has been localized on free ribosomes and not membrane-bound ribosomes. Free polysomes and membrane-bound polysomes, prepared by classical cell fractionation techniques from rat liver, were incubated for protein synthesis in a cell-free system derived from rabbit reticulocytes. Characterization of the total translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, as well as by immunoprecipitation with anti-rat albumin anti-serum, confirmed that good separation of the two polysome classes was achieved. Uricase and catalase were immunoprecipitable from translation products directed by free polysomes or phenol-extracted free polysomal mRNA but not from products of membrane-bound polysomes. Furthermore, unlike albumin, nascent uricase and catalase were not cotranslationally segregated by dog pancreas microsomal membranes. The results indicate that uricase and catalase are transferred to the interior of peroxisomes by a post-translational mechanism; an hypothesis is formulated here for the biogenesis of peroxisomes.

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) of Bacillus fastidiosus was purified to homogeneity in a two-step procedure and was crystallized. The native molecule had a molecular weight of 145 000--150 000 and was composed of subunits of two kinds (Mr = 36 000 and 39 000) in a 1 : 1 ratio. The quaternary structure of the enzyme was reversibly altered, with concomitant loss of activity, at temperatures between 40 and 60 degrees C. No evidence was found for the involvement of metal ions or coenzymes in the uricase reaction. The enzyme was inhibited by various metal ions and by cyanide. The isoelectric point of the enzyme was 4.3, and pH optimum 9.5 and the optimal temperature 30--35 degrees C. Only uric acid was oxidized by the enzyme and 9-methyluric acid, xanthine, 8-azaxanthine and oxonic acid were competitive inhibitors. Uricase synthesis was repressed by allantoin and allantoate, even in the presence of uric acid, which induced synthesis of the enzyme. Molecular oxygen was an important environmental factor in the control of uricase synthesis, probably due to its effect, as cosubstrate in the uricase reaction, in assessing the cytoplasmic concentration of allantoin. The highest amounts of uricase, up to half of the intracellular soluble protein content, was found in cells growing under limited oxygen supply in media containing uric acid as the main substrate.

In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569--571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.

A uricase producing strain, Candida utilis AS 2.117, was screened out and the uricase-forming conditions have been investigated. The results are obtained as follows: uric acid, xanthine and guanine were found to be effective inducers for enzyme formation. Corn steep liquor was essential for enzyme formation and cell growth. Sucrose, glucose, D-mannose and D-fructose were the suitable carbon sources for enzyme formation. To the medium containing corn steep liquor, addition of inorganic nitrogen sources was harmful to the enzyme formation, however, addition of organic nitrogen sources enhanced enzyme activity slightly. The suitable medium for uricase formation was consisted of sucrose 5%, corn steep liquor 3%, uric acid 0.1%, proteose-peptone 0.1%, biotin 0.05%, KCl 0.1%, NaCl 0.1% at pH 6.2. When the yeast was grown in 250 ml conic flask containing 30 ml of the medium mentioned above on the rotary shaker (200 r/min) at 25 degrees C for 21 h, uricase with 0.6 u/ml activity was obtained.