High-level production of uricase containing keto functional groups for site-specific PEGylation

We describe an E. coli-based optimized system for the production of uricase with keto functional groups incorporated efficiently and site-specifically. In the process, the orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair specific for p-acetylphenylalanine (pAcF) was optimized to be effective at pAcF incorporation, showing no toxicity to the host cells. The efficiency of pAcF incorporation was further improved by coupling five copies of the T-stem mutant suppressor tRNA gene omitted the 3′ terminal CCA with two constitutive copies of the D286R mutant aaRS gene in a single-plasmid construct. To assay the utility of the optimized system, we incorporated pAcF in response to three independent amber nonsense codons (Lys21TAG, Phe170TAG, Lys248TAG) into uricase. Under optimized expression conditions, 24 mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase (UOXWT). The desired specificity for incorporation of pAcF into uricase was confirmed. Kinetic measurements and spectroscopic study performed by CD did not show any relevant differences in the substrate affinity, the catalytic activity and protein secondary structure between native and mutant uricase. Additionally, the mutant uricase was site-specifically modified with methoxy-PEG-oxyamine (mPEG_5K-ONH₂). This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.     

Polyethylene terephthalate membrane as a support for covalent immobilization of uricase and its application in serum urate determination

A method is described for covalent immobilization of uricase onto polyethylene terephthalate (PET) membrane with a conjugation yield of 4.44 μg/cm² and 66.6% retention of initial activity of free enzyme. The enzyme exhibited an increase in optimum pH from pH 7.0 to 8.5 and K_m for uric acid from 0.075 mM to 0.13 mM but slight decrease in temp. for maximum activity from 37 °C to 35 °C after immobilization. A colorimetric method for determination of serum uric acid was developed using immobilized uricase, which is based on measurement of H₂O₂ by a color reaction consisting of 3,5-dichlorobenzene sulphonic acid (DHBS), 4-aminoantipyrine and peroxidase as chromogenic system. Minimum detection limit of the method was 0.05 mM. Analytical recovery of added uric acid (5 mg/dl and 10 mg/dl) was 94.3% and 89.8%, respectively. Within and between batch coefficient of variation (CV) were <3.2% and <4.3%, respectively. A good correlation (r = 0.98) was found between uric acid values by standard enzymic colorimetric method and the present method. The immobilized uricase was reused 100 times during the span of 60 days without any considerable loss of activity, when stored in reaction buffer at 4 °C. The support chosen for the present study was biocompatible, antimicrobial, inert, impact resistant, light weight and had good shelf life.     

Purification, and properties of a bovine uricase

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.     

Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid

Uricase was found to be stabilized by protamine from salmon testis. Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas free uricase was inactivated at 45 degrees C and showed activity over a broader pH range than free uricase. Automated analysis of uric acid was facilitated using the immobilized uricase. The standard curve for uric acid was linear in the range of 2 to 10 micrograms/sample and passed through the origin. This automated procedure was also applicable to the determination of uric acid in human serum. Protamine bound to glass beads is expected to be useful for the simple immobilization and stabilization of enzymes.     

假丝酵母尿酸酶形成条件

选出了一株产尿酸酶的产朊候丝酵母(Candida utilis)AS2．117。此菌株尿酸酶形成条件的研究表明：尿酸、黄嘌呤和鸟嘌呤对酶形成起诱导作用;玉米浆对菌株生长和酶形成起十分重要的作用;蔗糖、葡萄塘、D-甘露糖和果糖是酶形成的适合碳源;生物素对酶产生有促进作用;在含有玉米浆培养基中加入无机氮源对产酶无作用,添加有机氮略增加产酶量。尿酸酶形成最适培养基组成为(％)：蔗糖;,玉米浆3,尿酸0．1,蛋白胨0．1,生物素0．05,KCI0．1,NaCl 0.1。最适pH为6．2。在250ml三角瓶中装30ml培养基为最适。在200r／min的旋转摇床上25℃振荡培养21h,在此条件下最终酶活力可达0．6u／ml。     

Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4 s at pH 7.5 and 40 °C, when operated at 0.4 V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 μM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r = 0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4 °C.     

Production of a thermostable uricase by a novel Bacillus thermocatenulatus strain

A novel uricase-producing bacterium was identified based on its 16S rRNA sequence as Bacillus thermocatenulatus. The kinetic constants for this uricase, determined with uric acid as the substrate, were a V(max) of 0.99U/ml of enzyme and a K(m) of 0.25mM. After heat treatment at 75 degrees C for 45min, the uricase retained about 100% of its initial activity. The uric acid showed to be an inducer for uricase production. The effects of different factors on the enzyme production were studied. Pretreated cane molasses and corn steep liquor were the most promising carbon and nitrogen sources, respectively. When the strain was cultured at 30 degrees C at pH 7.0 for 30-36h, the uricase activity peaked at 1.25U/ml.     

Discrete analysis of serum uric acid with immobilized uricase and peroxidase.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.     

Role of uricase in the triggering of germination of Bacillus fastidiosus spores.

The likelihood that uric acid was the only compound capable of triggering germination of Bacillus fastidiosus spores was reinforced by the finding that ureidoglycollic acid, urea, NH4Cl, 2,8-dihydroxypurine and a combination of L-alanine and O-carbamoyl-D-serine were ineffective as germinants. Uric acid-triggered germination of B. fastidiosus was prevented by a range of inhibitors that also inhibited uricase activity in dormant spore extracts. O2 uptake during germination started immediately after addition of uric acid, possibly as a consequence of the oxidation of uric acid by the enzyme uricase. Germination showed a dependence on uric acid concentration, with a relatively high Km (4-5 mM). During the first 10 min of germination of heat-activated spores there was no detectable change in the number of spore-cortex reducing groups, indicating that selective cortex hydrolysis is not involved in the trigger mechanism of germination of B. fastidiosus. On the basis of the results, a model is proposed in which re-initiation of uricase activity is the mechanism by which B. fastidiosus spores are triggered to emerge from the dormant state.     

Regulation of expression of the Rhizopus oryzae uricase and urease enzymes

The regulation of intracellular urease and uricase activities was examined in Rhizopus oryzae. Urease activity (2.4 U/mg protein) was present in R. oryzae mycelium grown in minimal medium containing NH4CI as sole nitrogen source. This activity increased threefold under nitrogen derepression conditions, but no induction by urea was detected. Control of urease activity in R. oryzae differs from that found in Neurospora crassa but resembles the situation in Aspergillus nidulans. No uricase activity was detected in R. oryzae mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. Uricase activity was increased 10- to 40-fold under derepression conditions and was induced by exogenous uric acid (60- to 78-fold). Control of the R. oryzae uricase differs from that found in N. crassa and A. nidulans. This is the first analysis of the regulation of enzymes from the purine catabolic pathway in any member of the Zygomycetes.     

Allantoxanamide: A potent new uricase inhibitor in vivo

Allantoxanamide (2,4-dihydroxy-6-carboxamide-1,3,5-triazine) was studied as a uricase inhibitor in the rat. Uricase activity $\text{in vitro}$ was inhibited 50% by allantoxanamide at 9 × 10- M concentration. A single 250 mg/kg i.p. dose in the rat gave rise to a serum uric acid level of 14 mg/dl 6 hr after dosing; serum uric acid was still elevated (10 mg/dl) after 24 hr. At this dose level, deposition of uric acid in kidney tubules was observed. Studies with [8-¹⁴ C] uric acid indicated that the effect of allantoxanamide on serum uric acid was due to inhibition of uricase. The allantoxanamide-treated rat may serve as a useful animal model for the study of problems related to purine biosynthesis, drug-induced hyperuricemia and hyperuricosuria, and associated nephropathy.     

Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus

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⁺?>?Ca²⁺?>?Zn²⁺?>?Fe³⁺?>?Ni²⁺?>?Mg²⁺?>?Mn²⁺?>?Cu²⁺.     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

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.     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

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.     

尿酸酶－鲁米诺化学发光传感器

研制了依赖于鲁米诺化学发光反应和固定化尿酸酶柱的测定血清尿酸的生物传感器。其测定血清样品响应时间47s。测定每份样品需时1．5min,样品体积17μl。工作曲线的线性范围1~20mg／dl。批内不精密度3．22％～4.36％,批间6.18%～7．8%。测定值回收率为93％～109％。与医院常规酶试剂盘方法比较相关系数r=0.9909。固定化尿酸酶柱室温使用,4℃冰箱保存,连续使用5个半月测定样品2000次以上,仍保持原酶柱活力的94%。     

Observations on the regulation of uricase activity during development of Drosophila melanogaster

To elucidate the mechanisms involved in the regulation of uricase activity in Drosophila melanogaster, a comparative analysis of the patterns of uricase activity during development was undertaken for the wild type, Ore-R, and the mutants ry ² and ma-1. Uricase activity in ry ² and ma-l, unlike that in Ore-R, increased rapidly following emergence of the adult. This study indicates that uricase in Drosophila, in contrast to that in several microorganisms, is not induced by uric acid, since ry ² and ma-l with no detectable uric acid have higher activity than the wild type.This study was supported by grants from NIH, PHS 5-TO 1 GM 0071-13 and 1F02 HD 50,527-01     

Liver uricase in Camelus dromedarius: purification and properties

1. Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3) was purified 750-fold from the liver of Camelus dromedarius. 2. The enzyme is a tetramer with a Mr of 100,000, displays high specificity for uric acid with a Km of 12 microM and is inhibited by a selected number of purine derivatives carrying oxygen at the C2 position. 3. The effect of pH and the inhibition by thiol compounds and chelating agents on the enzyme activity is reported. 4. Some lines of evidence suggesting the possibility of interaction of camel liver uricase with oligonucleotides are presented.     

Modification of yeast uricase with polyethylene glycol: disappearance of binding ability towards anti-uricase serum

Uricase from Candida utilis was modified with activated polyethylene glycol (2-O-methoxypolyethylene glycol-4,6-dichloro-s-triazine) of molecular weight of 5,000 daltons. The modification of 43% of the total amino groups in the uricase molecule gave rise to a complete loss of the binding ability towards anti-uricase serum from rabbit. This modified uricase retained 15% of the enzymic activity of non-modified uricase.     

Postnatal development of hepatic uricase and peroxisomes in baby pigs

1. The activity of uricase, the densities of peroxisomes and cores in liver samples of baby pigs up to 4 weeks of age were investigated. 2. From 1 to 4 weeks of age, uricase activity as well as counts of cores and peroxisomes increased 5.5-, 3.3- and 2-fold. 3. Uricase activity and counts of cores and peroxisomes were correlated (P less than 0.001) with age in linear relationships. 4. Calculated for time of birth uricase activity was very low and ratio of cores to peroxisomes was 1:7. 5. From 0 to 28 days of age the calculated increases of uricase activity and counts of cores and peroxisomes were 178-, 10- and 3-fold.     

Development and analytical application of an uric acid biosensor using an uricase-immobilized eggshell membrane

An uric acid biosensor fabricated from a uricase-immobilized eggshell membrane and an oxygen electrode was presented. The detection schemes involve the enzymatic reactions of the uricase leading to the depletion of dissolved oxygen level upon exposure to uric acid solution. The decrease in oxygen level was monitored and related to the uric acid concentration. The scanning electron micrographs show the microstructure of the eggshell membrane within which the uricase is successfully immobilized. The effects of enzyme loading, pH, temperature, and phosphate buffer concentration on the response of the biosensor were investigated in detail. The uric acid biosensor has a linear response range of 4.0-640 microM with a detection limit of 2.0 microM (S/N=3). The response time was less than 100 s. The biosensor exhibited good repeatable response to a 0.10mM uric acid solution with a relative standard deviation of 3.1% (n=7). The reproducibility of fabrication of the biosensors using four different membranes was good with a R.S.D. of 3.2%. The biosensor showed extremely good stability with a shelf-life of at least 3 months. Some common potential interferents in samples such as glucose, urea, ascorbic acid, lactic acid, glycine, DL-alpha-alanine, DL-cysteine, KCl, NaCl, CaCl2, MgSO4, and NH4Cl showed no interferences on the response of the uric acid biosensor. The biosensor was successfully applied to determine the uric acid level in some human serum and urine samples, and the results agreed well with those obtained by a commercial colorimetric assay kit.