Studies on the Formation of Uricase by Streptomyces

A strain of Streptomyces sp. produced little of uricase in the cells when they were grown in a medium consisted of peptone, glucose and inorganic salts, even in the presence of urate. The cells, however, formed a large amount of the enzyme, when they were incubated with urate in K-phosphate buffer. The amount of uricase thus formed was maximum by the cells which were harvested at the middle logarithmic phase of the preliminary growth. The induced formation of uricase required K ions in addition to Mg ions and was accelerated by glucose and some other carbon sources. The enzyme formation was inhibited completely by chloramphenicol at a low concentration. An equimolar allantoin to urate decomposed by the cells was accumulated in the incubation mixture. More than 3.0 units of uricase per g of wet cells were produced under the best conditions known from the present experiments. The derepression of uricase formation in the resting cells incubated in the phosphate buffer was discussed.     

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.     

Studies on the Formation of Uricase by Streptomyces

The induced formation of uricase by the cultured cells of Streptomyces sp. and the effect of purine bases on the enzyme formation were studied. The microorganism was grown in media containing urate and/or purine bases (adenine, guanine, hypoxanthine or xanthine) and the development of the uricase activity of the cells were measured at intervals. The disappearance of urate and purine bases from the media was also determined. Without the purine bases, the production of uricase was significantly low even in the presence of urate and the disappearance of urate from the medium was in a slow rate. Upon the addition of hypoxanthine or xanthine in the presence of urate, a significant increase in the uricase activity of the cells and a concomitant rapid decrease of urate in the medium were observed. The purine bases added to the media were incorporated into the cells at a relatively early period of the culture and appeared to be converted into urate within the cells. The repression of uricase formation in the cultured cells and the derepression by the addition of the purine bases were discussed.     

Effect of Endogenous and Exogenous Urate on the Uricase Formation by Streptomyces sp.

Cells of a strain of Streptomyces sp. were incubated with an equivalent quantity of urate, xanthine, 6,8-dihydroxypurine or hypoxanthine in a medium deprived of other nitrogen source. The amount of uricase produced by these cells was shown to differ significantly, increasing in the following order of purine bases added to the medium: urate, xanthine, 6,8-dihydroxypurine and hypoxanthine. Of these was only urate indicated to be the inducer of uricase formation, and the difference in the quantity of uricase produced was found to be based on the duration of enzyme formation. The rate of uricase formation was essentially identical regardless of the purine bases supplied to cells.

Allantoin was accumulated in medium in remarkably different manners depending on the purine bases, which suggested the diversity in the mode of generation of urate in cells. Urate was generated at the slowest rate in the cells incubated with hypoxanthine, although the largest amount of uricase was produced, However, urate supplied to cells at the same rate but from medium failed to support the enzyme formation when the activity increased to a certain level. In order that the same amount of uricase was produced by the cells incubated with the different purine bases, the initial concentration of the purine bases should be raised so that they could remain in medium for the same incubation time.

Intracellular compartmentalization that might segregate endogenous and exogenous urate and might cause the difference in “effeciency” of these urate molecules as the inducer of uricase formation has been discussed.     

Uricase production by a recombinant <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> strain harboring <Emphasis Type="Italic">Candida utilis</Emphasis> uricase gene

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.     

Urate-mediated regulation of urate oxidase inChlamydomonas reinhardtii

Summary Expression of uncase (urate oxidase) fromChlamydomonas reinhardtii has been investigated by using specific polyclonal antibodies. By Western blot analyses performed under nondenaturing conditions, a 124 kDa protein band corresponding to active uricase was detected in protein extracts from cells cultured with urate or nitrogen-starved cells. This protein band was absent in cells cultured with ammonium. Besides the 124 kDa band, the antibodies also reacted with a massive protein band, with an apparent molecular mass of 500 kDa, that was detected in all nutritional conditions assayed. In vitro, inactive uricase from cells grown with ammonium was activated by incubation in presence of urate. The appearance of uricase activity in vitro coincided with a decrease of the 500 kDa protein and an increase of the 124 kDa band corresponding to the active enzyme. We suggest that a posttranslational regulation of uricase synthesis takes place inC. reinhardtii, and that urate may be responsible for the assembly or maturation of inactive precursors to form the active uricase.     

Studies on Yeast Uricase

A method for obtaining a highly purified preparation of yeast uricase was developed. The procedure included extraction of uricase from the uric ase-induced yeast cells, fractionation with ammonium sulfate and chromatography on a DEAE-cellulose column. The purified yeast uricase was shown to be ultracentrifugally homogeneous. The enzyme acted best at pH 8.5 and was stable in a range from pH 7.0 to 11.0 and at temperatures lower than 40°G. The Michaelis constant for urate was calculated to be 5.88 × 10^?6 m at pH 8.5, borate buffer. Activity and stability of the enzyme, however, were found to be significantly affected by the kind of buffer used. The enzyme was sensitive to heavy metal ions such as mercuric and cupric ions, but the sensitivity was influenced by the kind of buffer used.     

Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules

Nodulin-35, a protein specific to soybean root nodules, was purified under non-denaturing conditions (DEAE-cellulose followed by Sephacryl S-200 chromatography) to homogeneity. The holoprotein showed uricase (EC 1.7.3.3) activity. Analytical ultracentrifugation under non-denaturing conditions revealed a molecule of 124 kd, S°_20W = 8.1; however, under denaturing conditions a value of 33 kd, S°_20W = 1.9, was obtained. This indicated that nodulin-35 is the 33-kd subunit of a specific soybean root nodule uricase (uricase II) and that the enzyme contains four similar subunits. The native molecule contains ˜1.0 mol Cu²⁺ per mol, has an isoelectric point of ˜9.0 and a pH optimum for uricase activity at 9.5. Uricase activity found in young uninfected soybean roots is due to another form of enzyme (uricase I) which is of ˜190 kd, has maximum activity at pH 8.0 and does not contain any subunit corresponding in size to nodulin-35. Uricase I, also present in young infected roots, declines at a time when nodulin-35 appears. Monospecific antibodies prepared against uricase II (nodulin-35) showed no cross-reactivity. Uricase II was localized in the uninfected cells of the nodule tissue. These results are consistent with the concept that a nodule-specific ureide metabolism takes place in peroxisomes of uninfected cells, and suggest the participation of uricase II in this pathway.     

Studies on the xanthine oxidase activity of mammalian cells

Xanthine oxidase in man is confined to but a few tissues and is absent from cultured cell strains. In rodents, however, the enzyme is more widely distributed among the tissues and can be demonstrated in most cell lines. Rodents possess the enzyme uricase and are therefore able to carry purine catabolism one step further than man. Preliminary results suggest that uricase is restricted to but a few rodent tissues and is absent from cultured rodent cells. Hence it may be that in each species only the final enzyme of purine catabolism is tissue restricted. In other experiments, mammalian cells were grown in the presence of compounds known to induce xanthine oxidase in a eukaryotic fungus (Aspergillus nidulans). These compounds did not induce the enzyme in mammalian cells.Supported by program project grants 1-PO-GM 15419 and GM 18153-01, National Institutes of Health, United States Public Health Service.     

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.     

Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

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⁺.     

Developmental changes of adenosine deaminase, xanthine oxidase, and uricase in mouse tissues

The activities of adenosine deaminase, xanthine oxidase, and uricase were followed in the liver, kidney, stomach, and intestine during pre- and postnatal development of the mouse. Results indicated that some type of coordinate control exists between the uricase and the xanthine oxidase levels in liver, stomach, intestine, and kidney. No coordinate control was seen between adenosine deaminase and xanthine oxidase in liver and kidney. The developmental changes between the intestine and stomach xanthine oxidase and adenosine deaminase were found to be related. The results obtained were consistent with the idea that intermediate metabolites in a pathway play some role in controlling the level of enzymes further down the pathway.A superificial resemblance in the timing of changes in feeding habits and changes in enzyme levels during development was found. Results of artifical change of feeding habit indicate that the control of enzyme levels was inherent rather than the result of dietary change.     

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.     

Purification and characterization of uricase,a nitrogen-regulated enzyme,from Neurospora crassa

Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora. Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K_m value of uricase for uric acid was estimated to be 4.2 × 10^?5, m. Oxonic acid was shown to be a competitive inhibitor of uricase, with a K_i value of 6.7 × 10^?7, m. Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora. The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.     

PEG-重组酵母尿酸酶结合物的基本特性研究

重组Candida utilis尿酸酶由含PET-Uricase表达质粒的重组E.coli JM109(DE3)经乳糖诱导表达,菌体破碎后依次经过硫酸铵沉淀、阴离子交换层析和凝胶过滤层析可以获得纯度95%的重组尿酸酶。还原性SDS-PAGE和HPLC测得其亚基表观分子量和天然分子量分别约为33 kDa和130 kDa。获得的纯酶与20 kDa (mPEG)2 -Lys-NHS在特定的条件下反应合成PEG-重组酵母尿酸酶结合物,考察了重组酵母尿酸酶PEG化前后的基本性质,结果显示PEG化尿酸酶的最适pH为7.5,较修饰前下降了1个pH单位,酸碱稳定范围与修饰前类似,都在pH 6-10范围内稳定;修饰前后最适温度均为40℃,重组酵母尿酸酶的热稳定性和抗蛋白酶水解能力较PEG修饰前有较大提高;PEG化尿酸酶可保留修饰前酶活力的87.5%;在最适条件下,PEG-尿酸酶结合物的Km为3.57×10-5 mol/L,而修饰前测得的Km为3.91×10-5 mol/L。研究结果为深入探讨PEG化尿酸酶的结构与功能奠定了基础。     

A Review of: “Gel Permeation Chromatography,Eds. K.H. Altgelt and L. Segal Marcel Dekker,Inc., New York 1971 xvii + 646 pages ill., hard cover $24.75”

A simple, rapid procedure for the purification of uricase from mammalian tissue is reported. The procedure is based on the precipitation of mammalian uricase under certain dialysis conditions, and on its low solubility near neutral pH. Exceptionally high yields of homogeneous enzyme are obtained.     

Purification of uricase from mammalian tissue.

A simple, rapid procedure for the purification of uricase from mammalian tissue is reported. The procedure is based on the precipitation of mammalian uricase under certain dialysis conditions, and on its low solubility near neutral pH. Exceptionally high yields of homogeneous enzyme are obtained.     

Electrochemical detection of uric acid via uricase-immobilized graphene oxide

Measurement of the uric acid level in the body can be improved by biosensing with respect to the accuracy, sensitivity and time consumption. This study has reported the immobilization of uricase onto graphene oxide (GO) and its function for electrochemical detection of uric acid. Through chemical modification of GO using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) as cross-linking reagents, the enzyme activity of the immobilized uricase was much comparable to the free enzyme with 88% of the activity retained. The modified GO-uricase (GOU) was then subjected to electrocatalytic detection of uric acid (UA) via cyclic voltammetry (CV). For that reason, a glassy carbon electrode (GCE) was modified by adhering the GO along with the immobilized uricase to facilitate the redox reaction between the enzyme and the substrate. The modified GOU/GCE outperformed a bare electrode through the electrocatalytic activity with an amplified electrical signal for the detection of UA. The electrocatalytic response showed a linear dependence on the UA concentration ranging from 0.02 to 0.49 mM with a detection limit of 3.45 μM at 3σ/m. The resulting biosensor also exhibited a high selectivity towards UA in the presence of other interference as well as good reproducibility.