US3620923A - Urate oxidase and process for the production thereof - Google Patents

Urate oxidase and process for the production thereof Download PDF

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US3620923A
US3620923A US715623A US3620923DA US3620923A US 3620923 A US3620923 A US 3620923A US 715623 A US715623 A US 715623A US 3620923D A US3620923D A US 3620923DA US 3620923 A US3620923 A US 3620923A
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urate oxidase
solution
precipitated
uricase
further purified
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Pierre Laboureur
Marcel D P Brunaud
Claude Langlois
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APPLIC BOICHIMIQUES JOUJ EN JO
SOC D'ETUDES ET D'APPLICATIONS BOICHIMIQUES JOUJ-EN-JOSAS YVELINES AND ETAB LISSEMENTS CLIN-BYLA PARIS UGINE-KUHLMANN
Sanofi SA
CM Industries SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
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    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/815—Enzyme separation or purification by sorption
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S435/911—Microorganisms using fungi
    • Y10S435/939—Rhizopus

Definitions

  • the invention relates to a new enzyme or urate oxidase which is very active in promoting oxidation of uric acid to allantoin.
  • the urate oxidase is produced by fermentation under aerobic conditions of nutrient media containing sources of assimilable carbon, assimilable nitrogen and uric acid which have been seeded with suitable cultures of bacteria, fungi or yeasts. Isolation and purification of the urate oxidase is described.
  • This invention relates to a novel urate oxidase having a high activity (currently known by the name uricase), as well as to a process for the manufacture thereof.
  • uric acid is one of the principal products of the catabolism of purine bases and of the products which they contain, such as nucleic acids. It is also known that if catabolism does not take place or elimination of uric acid does not occur, this product can accumulate in the blood or the tissues and can be the cause of many disorders, especially of gout, certain forms of rheumatism, certain calculi in the region of the urinary system and various tissue changes, especially in the cardio-vascular system.
  • the uricase of the present invention is obtained from active strains of micro-organisms which are either bacteria, especially those of the genus Bacillus, or fungi especially those which belong to the genera Mucor, Rhizopus, Absidia, Fusarium, Alternaria, Penicillium, Aspergillus, Cephalosporium, Stemphylium and Macrosporum, or yeasts, especially of the genus Geotrichum. These genera belong to the orders of en bacteriales, actinomycetales, mucorales, moniliales, spheriales and endomycetales.
  • the uricase may also be obtained using bacteria of the genera Pseudomonas, Clostridium, Micrococcus and Bacterium, fungi of the genus Neurospora and yeasts of the genera Saccharomyces and Torula (Candida).
  • the invention comprises more particularly the production of the uricase using bacteria and fungi belonging to the species Streptomyces cellulosae and Strept. sulfureus, Bacillus megatherium, B. subtilis and B. cereus, Aspergillus flavus, Asp. oryzae, Asp. tamarii, Asp. terricola, Asp. luchuensis, Asp. niger, Asp. sydawi, Asp. nidulans, Asp. wentii, Asp. fonsecaeus, Asp. clavatus, Asp. ustus, Asp. Ierreus and Asp. ochraceus, Penicillium frequentans, Pen.
  • Pen. griseum Pen. canescens
  • Pen. spinulosum Pen. lhomii, Pen. waksmani, Pen. raistrickii, Pen. expansum, Pen. purpurescens, Pen. funiculosum, Pen. spiculisporum, Pen. veluu'num, Pen. purpurogenum, Pen. lilacinum, Pen. rubrum, Cephalosporium, Alternaria tenuis, Fusarium salani, Fus. moniliforme, Fus. coeruleum, Fus. axysporum and F us.
  • Suitable micro-organisms are given below. In this list each name is followed by the number assigned by the applicants to the strain in their collection and in brackets by the registered number of the strain deposited in the American Type Culture Collection (A.T.C.C.).
  • Bacteria Streptomyces cellulosae Krainsky 3 3 l 3( 21 184), Streptomyces sulphureus Krainsky 105(2[ 185 Bacillus megatherium of Barry 1552(21180), Bacillus megatherium of Barry l55l(2ll8l), Bacillus subtilis Cohn 522 l (2 l 18 3) and Bacillus cereus Frankland 1665(21182).
  • Fungi other than yeasts Aspergillus flavus Link 60l(20037), A.f. Link 602(20038), A.f. Link 603(20039), A.f. Link 604(20040), A.f. Link 605(20041), A.f. Link 606( 20042), A.f. Link 608(20043), A.f. Link 609(20044), A.f. Link 620(20045), A.f. Link 623(20046), A.f. Link 651(20056), Asp. niger, Van Tieghem 561(20057), Asp. sydowi Bainier and Sartory 661(20058), Asp.
  • the invention also includes a process for producing and purifying the urate oxidase thus making it possible to obtain the product in a very active form.
  • This activity expressed in uricase units, in accordance with a definition which will be given hereafter, is generally between 10 and 500 units per milligram. The method of measuring the activity is described subsequently.
  • a process for producing a urate oxidase which promotes the oxidation of uric acid to allantoin which comprises cultivating an active strain of a bacterium, a fungus or a yeast in or upon an aqueous nutrient medium comprising an assimilable source of carbon, a source of assimilable nitrogen, and uric acid under conditions of aerobic fermentation, separating the culture from the aqueous medium and isolating said urate oxidate from the culture and/or from the resulting aqueous medium.
  • the process of the invention involved cultivating the urate oxidase producing micro-organism, either by using a submerged pre-culture, sparsely or not sporulated, entirely in a liquid medium which is suitable agitated and aerated, making it possible to produce considerable amounts of the micro'organism, preferably at temperatures between 20 and 35 C. and during 4 to l2 days of cultivation, or by cultivation entirely upon a solid medium making it possible to obtain spores at temperatures between 20 and 35 C. during 6 to 15 days of cultivation.
  • the thus prepared inoculum is used to seed a nutrient medium containing urate ions and in effecting fermentation at a temperature which is preferably between 20 and 35 C.
  • the microbiological mass is separated from the liquid phase, preferably when maximum production has been attained, and the urate oxidase is extracted from the microbiological mass and/or from the liquid phase.
  • the crude urate oxidase thus obtained is then purified.
  • in preparing the inoculum precuitivation in depth may be carried out in a Czapeck-Dox or in a Sabouraud medium. If it is desired to use a solid medium, the culture may be effected upon malted rice.
  • Extraction of the endocellular urate oxidase from the microbiological mass may be effected by solidifying this mass, pulverizing it and submitting the pulverized material to a solidliquid extraction procedure.
  • a preferred strain of mycelium which is highly active is one obtained from an enriched culture of the genus Aspergillus and of the species flavus.
  • the process is applicable to other micro-organisms which produce endocellular urate oxidase in particular but not exclusively to those micro-organisms which have been set out above.
  • An analogous process may be used for those micro-organisms which produce exocellular urate oxidase, by treating the filtrates of the culture medium in the same manner as the cellular extracts of microorganisms which produce the endocellular urate oxidases.
  • the culture media should contain all the organic and inorganic constituents which are necessary for satisfactory development of the micro-organism and for the production of uricase in high yield. They should contain: (a) a source of assimilable carbon, in particular but not exclusively simple sugars such as glucose or saccharose, alone or in admixture, in concentrations between one and percent by weight (with respect to the total weight of the culture medium); (b) a source of assimilable nitrogen which is usually supplied in the form of inorganic nitrogen, such as ammonium chloride or sulfate, or an alkali nitrate, in concentrations between one and 10 percent by weight; and (c) inorganic elements such as phosphorus, magnesium, potassium, sodium, calcium and iron, in concentrations generally between 0.0001 and 0.5 percent by weight, as well as uric acid, in an amount between 0.01 and 0.2 percent by weight as an inductor.
  • a source of assimilable carbon in particular but not exclusively simple sugars such as glucose or saccha
  • oligodynamic substances such as amino acids, vitamins and growth factors, nucleotides and inorganic trace elements such as manganese, molybdenum, copper, cobalt and zinc.
  • these substances may be supplied, for example, by yeast extracts, malt extracts or by the hydrolysis products of animal and/or vegetable proteins or by inorganic salts. These may be used in concentrations between 0.0001 and 0.5 percent by weight.
  • the various constituents can be added either partially or entirely when preparing the culture medium, prior to seeding, or during the course of the fermentation and, in the case of certain elements, on account of their special importance, during the synthesis of the enzyme.
  • the constituents are essentially, but not exclusively, nitrogen and phosphorus-containing constituents and uric acid.
  • Cultivation is generally effected in depth employing agitation and aeration. Agitation may be by stirring which may vary, depending upon the installation used, between 10 and 200 r.p.m. Aeration is preferably effected at a rate between 0.05 and 0.5 liters of air per liter of medium per minute. Sufficient agitation can be achieved by bubbling through air. It should be noted however that agitation and aeration conditions which lead to too rapid and too intensive development of the mycelium may be prejudicial to optimum production of the enzyme.
  • DURATION OF THE CULTIVATION Cultivation is continued, preferably, until optimum enzyme activity is attained, this being determined by the analytical method which will be subsequently described. It is recommended that the fermentation be carried out to a substantially constant value of optimum enzyme activity, rather than to a value, which is possibly greater but is transitory.
  • the culture generally attains maximum activity after from to 60 hours culture; this time varies with the nature of the culture medium, the strain of inoculum employed and the conditions of cultivation.
  • the culture medium is filtered in order to separate the mycelium; the latter is washed with water and thereafter pulverized for the purpose of extracting the uricase.
  • pulverization it is possible to freeze the mycelium at low temperatures by means of carbon dioxide ice or carbon dioxide snow or by any other process which makes it possible to freeze the mycelial mass rapidly and completely.
  • freezing is carried out at temperatures between l5 and 30 C.
  • the mycelium is thereafter pulverized in an apparatus which enables complete separation of the mycelial constituents, without modifying the enzyme.
  • the pulverized product is extracted in water containing a buffer agent or in aqueous ammonia, brought to a pH which is preferably between 7 and 10, preferably 8.5, for the purpose of extracting the enzyme. This operation is carried out at a temperature below 30 C. by using a suitable extraction process. Moreover, in order to render the extract more stable a complexing agent may be added in a concentration of the order to 0.001 M, for example ethylenediamine tetraacetic acid, generally in the form of the sodium salt. The suspension is thereafter filtered and dried.
  • the mycelial extract thus obtained contains the uricase together with numerous proteins and other substances, in solution or in a colloidal state; they may have to be removed by desiccation or by means of salts with which they form complexes.
  • Suitable salts include calcium salts, such as calcium chloride and lead salts such as lead acetate, which make it possible to eliminate a great number of impurities in the form of insoluble compounds.
  • an antibiotic such as chloramphenicol, or an antiseptic, for example sodium azide, in amounts which may be between 10 and 500 ucgJmL, preferably cg/ml.
  • the uricase being soluble in water but insoluble in organic solvents and in concentrated aqueous solutions of inorganic salts such as ammonium sulfate, can be recovered easily, either by precipitation with the aid of an organic liquid which is miscible with water, for example ethanol, methanol or isopropanol or more preferably acetone, used in the proportion of 0.5 to 3 volumes per volume of extract, or by salting out with a water-soluble salt especially with a saturated solution of ammonium sulfate at room temperature, also used in a proportion of 0.5 to 3 volumes per volume of extract.
  • an organic liquid which is miscible with water for example ethanol, methanol or isopropanol or more preferably acetone
  • the mycelial extract may first be concentrated, by carrying out the operation under reduced pressure at a temperature below 40 C.
  • the precipitate thus obtained is separated, for example by centrifuging.
  • the organic liquid which it contains is removed rapidly under reduced pressure, without applying heat.
  • the precipitate can then be dissolved in water for the purpose of lyophilization, after filtration or centrifuging, which results in the production of a semipurified product.
  • filtration using membranes of a filter candle is carried out prior to lyophilization.
  • the salts may be removed either by dialysis or by rapid filtration using a suitable molecular sieve, for example, a dextran gel or a polyacrylamide gel.
  • the purification can be carried still further by subjecting the thus treated products to chromatography, which may be a cyclic or noncyclic process, by making use of columns of substances which make it possible to eliminate those impurities, in particular proteins, which are still present in the extract.
  • the substances which can be used for this purpose include columns of cellulose ion exchange materials, dextrans and polyacrylamides. Elution may be effected by means of liquids in which there is a continuous or discontinuous change in the pH or in the molarity thereof.
  • the uricase is obtained in the fortn of an amorphous, colorless or slightly yellow powder which is soluble in water and the conventional buffer solutions, the solubility being greater in alkaline solution than in an acid solution. It is insoluble in the usual organic solvents such as ethanol, methanol, acetone, diethyl ether and chloroform.
  • the pH of solutions having optimum activity is 8.5 and the temperature at which the uricase exhibits optimum activity is 30, the activity is, however, still quite high at 37 C.
  • the activity of the uricase is partially inhibited by the following cations: Zn, Cd, Mn", Fe Co, Ni, Al at concentrations of and 10" M, and by Hg at a concentration of 10 M.
  • Cu is a weak inhibitor (30 percent inhibition at 10" M). Calcium, sodium, potassium, magnesium and ammonium ions either do not or only slightly inhibit activity up to concentrations of 10" M.
  • borate, carbonate, acetate, citrate, nitrate, chloride and phosphate have no effect on the uricase at concentrations of 10 to 10' M; however the nitrate and chloride anions can become partial inhibitors at concentrations of the order of 10'" M.
  • the uricase has no effect upon caffeine, theobromine, theophylline, xanthine, 8-chloroxanthine or 2,8-dithio-6- hydroxypurine, that is to say, upon the principal biological products the chemical structure of which is closely similar to that of uric acid. Consequently this enzyme can be regarded as extremely specific.
  • the culture medium was sterilized for 30 minutes at 120 C. Seeding after cooling with the inoculum described above. Cultivation: during cultivation the temperature of the culture medium is maintained at 28 :l" C., the culture medium is aerated by means of sterile air blown in to the bottom of the container at a rate of 0.3 liters per liter of culture medium per minute. After 24 hours of cultivation under the indicated conditions kg. of mycelium having a content of l50 u./g. of
  • uricase is obtained.
  • the mycelium is washed with 40 liters of sterile distilled water and then cooled rapidly to -20 C.
  • the mycelium is frozen at 20 C., pulverized and then taken up in 40 liters of aqueous ammonia having a pH 9; after concentrating the solution to one-quarter of its original volume under reduced pressure and precipitation by addition of two volumes of acetone, 380 g. of a crude product containing 7,000 u./g. of uricase is obtained. This product is taken up in 3.8 liters ofa 0.002 M aqueous sodium carbonate solution. After centrifuging the solution is treated with an equal volume of a saturated aqueous solution of ammonium sulfate.
  • the precipitate thus obtained is separated by centrifuging, then taken up in an aqueous solution of 0.002 M sodium carbonate; the insoluble remainder is eliminated by centrifuging.
  • the supernatent solution is then freed from inorganic constituents using a column of a dextran gel buffered with 0.002 M sodium carbonate, and the active eluates are sterilized by passing them over a Millipore GS membrane and are then lyophilized. 57 g. of a product containing 49,000 u./g. of un'case are thus obtained.
  • lnoculum This is prepared by seeding 2 liters of a Saboraud liquid medium with an agar culture on a Czapeck-Dox culture medium. After 40 hours cultivation this culture is used for inoculating liters of a fermentation liquid containing the same culture medium as in example 1. At the end of 30 hours cultivation at 28 C. this second inoculum is used for seeding the actual fermentation medium.
  • Culture medium
  • Glucose as used in Example l 100 kg. Saccharose l5 kg. Sodium nitrate 6 kg. Dipotassium hydrogen phosphate 2.250 kg. Monopotassium dihydrogen phosphate 0.750 kg. Calcium carbonate 3 kg. Magnesium sulfate 7 H,O l kg. Ferrous sulfate 7 H,O 3 g. Soluble extract of maize l kg. Malt extract l kg. Casein hydrolysate 0.300 kg. Copper sulfate I g.
  • the culture medium was sterilized for l hour at C. Seeding, after cooling, was carried out with the inoculum produced as described above. Cultivation: During cultivation the temperature of the culture medium is maintained at 30il C. and the culture medium is agitated by means of a propeller revolving at r.p.m. The culture medium is aerated by means of sterile air at the rate of 0.2 liters/liter of culture medium per minute. After 31 hours of cultivation there is added to the culture medium 2 kg. of sodium nitrate, 0.5 kg. of yeast autolysate and 0.5 kg. of uric acid, all of which have been previously sterilized. After 43 hours of cultivation 60 kg.
  • mycelium containing I40 u./g. of uricase is obtained.
  • the mycelium is washed with sterile distilled water and then rapidly cooled to 20 C.
  • the frozen mycelium is pulverized and then extracted by means of 120 liters of aqueous ammonia having pH 9.
  • the pH of the extract is readjusted to 9 and the volume of this extract reduced to 40 liters by concentration under reduced pressure.
  • the concentrated product is purified by treatment with an aqueous solution of basic lead acetate containing 10 percent by weight of lead. After centrifuging the supernatent liquid which contains the uricase is admixed with 48 liters of a saturated aqueous solution of ammonium sulfate.
  • the precipitate obtained is separated by centrifuging, taken up in 2.5 liters of a 0.002 M aqueous sodium carbonate solution and then passed through a column of a dextran gel.
  • the active eluates are lyophilized and there is thus obtained 54 g. of a product containing 91,000 u./g. of uricase.
  • EXAMPLE 3 The process described in example 2 is repeated and there is obtained 58 g. of a product containing 87,000 u./g. of uricase. This product is dissolved in a 0.01 M ammonium carbonate solution and the solution, buffered by a 0.01 M aqueous solution of ammonium carbonate at pH 9 is passed over a column of diethylaminoethyl cellulose. The column is subsequently washed with a 0.05 M ammonium carbonate solution. The uricase is eluted from the column by increasing the molarity of the buffer solution during the course of the elution. There is obtained in liquid form the equivalent of 4.7 g. of a product containing 340,000 u./g.
  • the uricase of this invention renders possible the rapid, specific and intensive elimination of uric acid and of urates present in the animal organism by conversion into water-soluble allantoin. It can be used, in particular, as a laboratory product in research and analytical studies of uric acid.
  • the activity is measured by determining the quantity of uricase required, under conditions hereafter described, to oxidize one half of the uric acid present in the reaction medium. The determination is based upon the fact that the optical density of a solution of uric acid falls after incubation with the enzyme preparation. Uric acid in acid solution has a maximum absorption in ultraviolet light at 285 millimicrons. The optical density under the conditions of the determination is proportional to the concentration of the uric acid and this fact makes it possible to measure the uric acid utilized and, consequently, to determine the activity of the enzyme.
  • the unit of uricase activity is the quantity of enzyme which brings about the destruction of one-half of the quantity of the substrate, that is of the uric acid introduced initially under the conditions of the determination.
  • Tris tris-(hydroxymethyl)aminomethane
  • Tris Tris
  • 373 mg. of Complexon III are dissolved in 50 ml. of distilled water, the two solutions being admixed and made up to 500 ml. with distilled water;
  • the preceding primary buffered solution having a pH 8.5 is diluted tenfold (solution to be used in the daytime only).
  • the concentration is adjusted to about 0.5 u./ml.
  • the culture tubes are placed upon a water bath and after a preincubation period of 5 minutes 1 ml. of the uric acid solution containing 100 neg/ml. is introduced into the culture tubes 2 and 4.
  • the culture tubes are then left to incubate for 10 minutes at 30 C. and the reaction is stopped by adding 0.2 M hydrochlo ric acid.
  • Culture tubes 2 and 4 are respectively compared with tubes 1 and 3, which are used as controls, by means of an ultraviolet spectrophotometer using a wavelength of 285 mu.
  • Tubes l and 2 can be used as controls for a series of determinations of optical density. Tubes 3 and 4 are used for the actual determinations.
  • I C Calculating the results A standard curve for a uricase having a concentration equivalent to 50 or 100 u.lmg. is used. This curve was prepared from a fuller determination using four dilutions of the enzyme. On this standard curve the enzyme concentrations are recorded as abscissae and the optical densities obtained after the action of the uricase present in the diluted solutions are shown as ordinates. On the curve there is noted the optical density corresponding to the test and from the abscissa there is read off the dilution of the standard product which produces an activity equivalent to 2 ml. of the diluted solution used. A rapid calculation makes it possible to obtain the activity of the solution or of the initial product.
  • EXAMPLE Standard Product containing 50 u./mg. Concentration of the standard solution utilized is g.lml.:0.5 u./ml.
  • optical density obtained after the action of 2 ml. of the diluted solution is equal to 0.500. This optical density corresponds upon the standard curve to the action of 1 ml. of standard solution.
  • the uricase can be lyophilized in a sterile medium under conventional conditions using an aqueous solution which has been sterilized by use of a sterilizing filter such as a Millipore filter.
  • FIGS. 1 (intraperitoneal administration of the uricase) and 2 (intravenous administration) of the drawings illustrate these results in the form of two graphs, the reduction in the uricemia, expressed as a percentage of its original value being represented as ordinates, whereas the time in hours is represented as abscissae.
  • the uricase has been administered to dogs. However, as the dog does not have any uric acid in its blood, an investigation of the uricase activity in the plasma was carried out by injecting the animals with the uricase. It was found that the enzyme persists in the blood for more than 24 hours, irrespective of whether it is administered intravenously, intramuscularly or subcutaneously.
  • FIGS. 3 to 6 of the drawings In which the time in hours is shown on the abscissae (logarithmic scale) and in which the number of units of uricase activity per ml.
  • F IG. 6 of the drawings represents the action of the uricase (I 10 u./kg.; intravenous) on the uricemia in dogs excluding the liver circulation; the uricemia, expressed in peg/ml, is shown as ordinates and the time, expressed in minutes, is shown as abscissae.
  • the uricase does not have any pronounced effect in dogs upon blood pressure, cardiac frequency, cardiac electrogenesis, tensional responses to various chemical mediators or upon the respiratory movements. It has no effect in dogs on water or inorganic diuresis.
  • the uricase being a protein by nature behaves as an antigen. In the course ofthe various tests carried out in studying the antigenic effect of this product, it did not exhibit any allergic properties but only immunizing properties.
  • the uricase has no acute toxicity.
  • a dose of 10,000 u./kg. injected intravenously into mice causes no pathological symptoms.
  • Toxicity tests have been carried out upon dogs using dosages of 200 and 1,000 units per animal per day.
  • the uricase has been administered intravenously for 4 months without producing any abnormal phenomenon or noticeable histological modifications in the animals.
  • Table No. 1(8) sets out the results obtained. Twenty-four hours after treatment the extensive diminution ofthc uricemia observed in test No. I is also found to occur here. As a corolla ry the urinary allantoin increases very markedly.
  • Ui'ieumia Uricuiiiiii Days of Dosage Urinary eg. uric l i'iiiai'y eg. iii'ii' treat- (units) Diuiosis iilliintoiii acid/nil. Dinrvsis :illiintoiii tlt'lll/llll. 1 ⁇ ' (iiil./2 1 hr.) ling/'34 lll'.) pliisiiiii) tiiil.2t 111'.) (iiigJl-l hr.) pliisiiizi) 1,000 2, 650 G5. 1 3, 200 54. 7 40.
  • the uricase can be administered parenterally, notably by the intravenous, subcutaneous or intramuscular routes, or by oral or endorectal administration.
  • compositions for intravenous administration are that of an aqueous solution which contains, at the time of application, 1,000 units of lyophilized and sterilized uricase and 5 ml. of a sterilized solvent having the following formula:
  • genus selected from the group consisting of Mai-0r, Rhi'zupur,
  • Distilled water in an amount sufficient for 1,000 ml. 55 Absidia, Fusarium, Allemari'a, PtIIlCU/illfll. Aspt'rgillus.
  • the lysophilization of the uricase was carried out in a sterile cellhalwpmiumr """P P a medium under the usual conditions, using an aqueous solution Gmmdmm' D which had been sterilized on a sterilizing filter such as the Mil- The process?
  • the urate oxidase is by storing the solutions at a sufficiently low temperature
  • the Preclp'mted from j' by the addi' solutions being prepared from the highly purified uricasc and non thereto of an organic l quid which is miscible with water from an aqueous solution of ammonium carbonate having a and the I concentration of 0.1 M ammonium ions, this solution having The process cla'med m clam 3 m wh'ch mdasc been brought PH 8 by means of carbon dioxide
  • a Soluion is precipitated from the resulting aqueous solution by the addicontaining, for example, 200,000 units of uricase pm 000 ion thereto of aconcentrated solution of a water-soluble salt.

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3767533A (en) * 1970-12-26 1973-10-23 Noda Inst For Scientific Res Process for producing uricase
US4062731A (en) * 1976-07-21 1977-12-13 Eastman Kodak Company Production of uricase from micrococcus luteus
US4064010A (en) * 1976-07-21 1977-12-20 Eastman Kodak Company Purification of uricase
US4394450A (en) * 1982-03-01 1983-07-19 Miles Laboratories, Inc. Method for purification of uricase
FR2664286A1 (fr) * 1990-07-05 1992-01-10 Sanofi Sa Nouvelle souche d'aspergillus flavus et son utilisation pour la production d'urate-oxydase.
WO1994001139A1 (en) * 1992-07-13 1994-01-20 Baylor College Of Medicine Targeting somatic gene therapy to joints
US5792751A (en) * 1992-04-13 1998-08-11 Baylor College Of Medicine Tranformation of cells associated with fluid spaces
US20100255100A1 (en) * 1997-12-31 2010-10-07 Altus Pharmaceuticals Inc. Stabilized protein crystals, formulations comprising them and methods of making them

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6031472B2 (ja) * 1978-12-14 1985-07-22 協和醗酵工業株式会社 酸性ウリカ−ゼ
IT1141061B (it) * 1980-09-19 1986-10-01 Anic Spa Procedimento per la produzione di uricasi
JPH0671425B2 (ja) * 1985-06-05 1994-09-14 サッポロビール株式会社 ウリカ−ゼおよびその製造法
NZ234453A (en) * 1989-07-13 1993-01-27 Sanofi Sa Recombinant dna encoding urate oxidase, and vector, host, protein and pharmaceutical compositions associated therewith
US5382518A (en) * 1989-07-13 1995-01-17 Sanofi Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells
FR2656530B1 (fr) * 1989-12-29 1994-09-23 Sanofi Sa Gene recombinant pour une expression dans les cellules eucaryotes d'une proteine telle que l'urate oxydase.
FR2649720A1 (en) * 1989-07-13 1991-01-18 Sanofi Sa Recombinant gene which encodes a protein such as urate oxidase
FR2656531B1 (fr) * 1989-12-29 1992-04-24 Sanofi Sa Promoteur artificiel pour l'expression de proteines dans le levure.
US5407822A (en) * 1991-10-02 1995-04-18 Sanofi Artificial promoter for the expression of proteins in yeast
US6783965B1 (en) * 2000-02-10 2004-08-31 Mountain View Pharmaceuticals, Inc. Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates
AU770014B2 (en) * 1998-08-06 2004-02-12 Duke University Peg-urate oxidase conjugates and use thereof
US9441210B2 (en) 2013-06-26 2016-09-13 Food Industry Research And Development Institute Method of reducing levels of uric acid

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3431176A (en) * 1966-02-02 1969-03-04 Toyo Boseki Preparation of uricase
US3475276A (en) * 1965-07-28 1969-10-28 Ono Pharmaceutical Co Method of producing uricase from yeast

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3475276A (en) * 1965-07-28 1969-10-28 Ono Pharmaceutical Co Method of producing uricase from yeast
US3431176A (en) * 1966-02-02 1969-03-04 Toyo Boseki Preparation of uricase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Colowick et al., editors, Methods in Enzymology Vol. I, pages 56 61 (1955) *
Kida et al., (I) Journal of Fermentation Technology Vol. 44 No. 11 pgs. 789 796 (1966). *
Kida et al., (II), Journal of Fermentation Technology Vol. 44 No. 11 pgs, 797 804. (1966). *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3767533A (en) * 1970-12-26 1973-10-23 Noda Inst For Scientific Res Process for producing uricase
US4062731A (en) * 1976-07-21 1977-12-13 Eastman Kodak Company Production of uricase from micrococcus luteus
US4064010A (en) * 1976-07-21 1977-12-20 Eastman Kodak Company Purification of uricase
US4394450A (en) * 1982-03-01 1983-07-19 Miles Laboratories, Inc. Method for purification of uricase
FR2664286A1 (fr) * 1990-07-05 1992-01-10 Sanofi Sa Nouvelle souche d'aspergillus flavus et son utilisation pour la production d'urate-oxydase.
US5792751A (en) * 1992-04-13 1998-08-11 Baylor College Of Medicine Tranformation of cells associated with fluid spaces
WO1994001139A1 (en) * 1992-07-13 1994-01-20 Baylor College Of Medicine Targeting somatic gene therapy to joints
US20100255100A1 (en) * 1997-12-31 2010-10-07 Altus Pharmaceuticals Inc. Stabilized protein crystals, formulations comprising them and methods of making them
US8558048B2 (en) 1997-12-31 2013-10-15 Althea Technologies, Inc. Stabilized protein crystals, formulations comprising them and methods of making them

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GB1198764A (en) 1970-07-15
SU421162A3 (ru) 1974-03-25
BE712846A (enrdf_load_stackoverflow) 1968-07-31
ES352030A1 (es) 1969-07-01
DK127333B (da) 1973-10-22
FR6301M (enrdf_load_stackoverflow) 1968-09-09
CS153458B2 (enrdf_load_stackoverflow) 1974-02-25
NL6804365A (enrdf_load_stackoverflow) 1968-09-30
FR1529675A (fr) 1968-06-21
US3810820A (en) 1974-05-14
JPS5313704B1 (enrdf_load_stackoverflow) 1978-05-12
DE1642656A1 (de) 1971-05-19

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