MN-SUPEROXIDE DISMUTASE, ITS PRODUCTION AND USE

Abstract: A novel superoxide dismutase is produced by cultivat­ ing a microorganism belonging to the genus Serratia. The superoxide dismutase is useful as an antiinflammatory agent.

Description: SUPEROXIDE DISMUTASE, ITS PRODUCTION AND USE This invention relates to superoxide dismutase, its production and use. The present inventors undertook a diligent research for discovering a new superoxide dismutase and found that a microorganism belonging to the genus Serratia was able to produce such a new superoxide dismutase. This finding was followed by a further series of studies which have resulted in the present invention described in detail hereinafter. This invention is directed to: (1) a superoxide dismutase having a defined set of properties, (2) a method for producing said superoxide dismutase characterized by cultivating a superoxide dismutase-producing microorganism belonging to the genus Serratia in a culture medium to thereby cause said microorganism to elaborate and accumulate superoxide dismutase in said culture medium and harvesting said superoxide dismutase from the resulting cults broth, and (3) an antiinflammatory agent which contains an effective amount of superoxide dismutase having a defined set of properties as an active ingredient in association with a pharmaceutically acceptable carrier or excipient therefor. The microorganism employed for production of the contemplated substance of this invention is any of the microorganisms belonging to the genus Serratia and capable of producing superoxide dismutase, inclusive of a mutant strain thereof. Among such microorganisms there are mentioned Serratia liquefaciens, Serratia marcescens, Serratia marinorubra, and concretely mentioned Serratia liquefaciens IFO 12979, Serratia marcescens IFO 3046, Serratia marcescens IFO 3759 (ATCC 4002), Serratia marcescens IFO 3736, Serratia marcescens E-15 ATCC 21074, Serratia marinorubra IFO 12973 (ATCC 27614). The above microorganisms designated by IFO numbers are listed up in Institute for Fermentation Osaka List of Cultures 1978 Sixth Edition, published by Institute for Fermentation, Osaka, Japan, and the microorganisms designated by ATCC numbers are listed up in the American Type Culture Collection Catalogue of Strains I Fourteenth Edition 1980 published by American Type Culture Collection, Rockville, Maryland, U.S.A. Microorganisms of the genus Serratia, in general, are liable to undergo change in characteristics and can be easily mutated by means of artificial treatments such as irradiation with X-rays, ultraviolet rays or radiation, mutagenic agents (nitrosoguanidine, ethyleneimine, etc.). Any of the resulting mutants can be employed for the purposes of this invention unless they have lost the ability to produce the superoxide dismutase. The production of the contemplated superoxide dismutase is carried out by cultivating said microorganism in a culture medium and harvesting and purifying the resulting elaboration product, which is the desired product, from said culture medium. The culture medium may be liquid or solid, only if it contains nutrient sources which the particular strain of microorganism can utilize, although a liquid culture medium is desirable when mass production is contemplated. Incorporated in such a medium are sources of nutrients which the microorganism can assimilate, digest or otherwise utilize, such as carbon sources, nitrogen sources, inorganic materials, trace nutrients and so on. The carbon sources may for example be starch, dextrin, glucose, sucrose, etc., and the nitrogen sources may for example be corn steep liquor, soybean meal, extracted soybean meal, yeast extract, denucleated yeast, casein, cotton seed meal, nitrogen-containing compounds such as ammonium chloride, ammonium sulfate, etc. The inorganic materials include, for example, sodium salts (e.g. sodium chloride), phosphates (e.g. sodium phosphate), calcium salts (e.g. calcium carbonate), potassium.salts (e.g. potassium chloride, potassium phosphate), magnesium salts (e.g. magnesium sulfate), manganese salts (e.g. manganese chloride, manganese carbonate), iron salts (e.g. iron chloride), zinc salts (e.g. zinc carbonate) and so on. The trace nutrients may for example be vitamins (e.g. vitamin B1, B2), fatty acid esters [e.g. esters (e.g. methyl or ethyl esters) of oleic acid, lauric acid, caproic acid, etc.)], nucleic acids (e.g. ribonucleic acid, deoxyribonucleic acid), related compounds of nucleic acids (e.g. inosinic acid, guanylic acid, adenylic acid) and so on. For defoaming purposes, oils (e.g. soybean oil, corn oil, peanut oil), synthetic antifoams [e.g. Tween 20, 60 and 80 (Kao-Atlas Co. Ltd., Japan), Actocol (Takeda Chemical Industries, Ltd., Japan)], etc. may also be incorporated. The cultivation Er^ > thod may for example be stationary culture, shake culture :-r aerated stir (submerged aerobic) culture. Submerged aerobic culture, in particular, is especially useful for production-scale runs. While cultural conditions vary with the condition and composition of medium, strain of microorganism, cultural method, etc., cultivation is desirably conducted at about 20 to 400C for about 10 to 100 hours, preferably, about 20 to 72 hours, the pH of the medium being about 4 to 9 and, preferably, about 6 to 8. The desired enzyme is mostly accumulated intracellularly. This enzyme is harvested and purified by conventional purification procedures. It is advantageous to collect the cells from the culture broth by centrifugation, filtration or Ool.le other known separatory mcthod, disru!,t the cells by such a procedure as ultrasonication, grinding with glass beads, or a method involving the use of a surfactant or solvent, and extract the enzyme from the disrupted mass. In harvesting the enzyme, the same or an other known separate: and purification procedure in the field of the art can be followed in any case to obtain a product of optional purity. Thus, the enzyme-containing disrupted mass is centrifuged or filtered to give a supernatant or filtrate. To this enzyme extract is added ammonium sulfate or acetone, and the resulting precipitate is collected by centrifugation, dissolved in a small quantity of water, desalted by dialysis and lyophilized, whereupon a crude enzyme powder is obtained. This crude enzyme powder can be purified by means employed conventionally for purification of enzymes. By way of illustration, gel filtration with Sephadex (Pharmacia Fine Chemicals AB, Sweden), column chromatography on diethylaminoethyl-cellulose (Serva Fine Biochimica, West Germany) or a like procedure can be followed to isolate superoxide dismutase. In this manner, superoxide dismutase can be separated and isolated in a desired purity. The enzymological and chemical properties of the superoxide dismutase obtained in Example 2 which appears hereinafter are as follows. (a) Activity: It dismutates a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule. (b) Substrate specificity: It acts on superoxide ion. (c) Optimum pH range: The optimum pH range of this enzyme is about 7 to 8. Fig. 1 shows a pH-activity curve of this enzyme. (d) Stable pH range: The enzyme is stable in the pH range of about 6 to 11. Fig. 2 shows a pH-stability curve of this enzyme. (e) Optimum functional temperature: About 20 to 450C. (f) Thermostability (conditions of inactivation): The enzyme was treated at pH 7.8 and various temperatures for various time periods. The enzyme is stable at 370C for 60 minutes but is inactivated about 50% when treated at 50 to 600C for 60 minutes and almost 100% when treated at 800C for 5 minutes. Fig. 3 shows a thermostability of this enzyme. (g) Molecular weight: As determined by the gel filtration method using a Sephadex G-100 column [The Biochemical Journal 91, 222-233, 1964], this enzyme has a molecular weight of about 4.8 x 104. When the enzyme is first treated with 1% sodium laurylsulfate (SDS) and then subjected to polyacrylamide gel electrophoresis in the presence of the SDS [The Journal of Biological Chemistry 244, 5074-5080, 19691, the enzyme is 4 found to have a molecular weight of about 2.4 x 10 These values indicate that this enzyme is a dimer consisting of subunits each having a molecular weight of about 2.4 x 104. (h) Elemental analysis: C = 48.90+2.08; H = 7.05+2.0%, N = l4.80i2.0% (i) Absorptions in the ultraviolet and visible regions of th spectrum: Fig. 4 shows an ultraviolet absorption spectrum of this enzyme and Fig. 5 shows a visible absorption spectrum of this enzyme. In the W region, this enzyme gives an absorption maximum at about 280 nm, an absorption minimum at about 253 nm and a shoulder at about 290 nm. The absorbance ratio (260 nm/280 nm) is 1.8. In the visible region, the enzyme shows an absorption maximum at about 470 to 475 nm, an absorption minimum at 375 to 380 nm and a shoulder at about 610 nm. (j) Infrared absorption spectrum: Fig. 6 shows an infrared absorption spectrum of this enzyme measured by the KBr method. Characteristic absorptions are 3.02 (s), 3.38 (m), 6.00 (s), 6.60 (s), 6.95 (m), 7.25 (m), 8.10 (m), 8.50 (w), 9.10 (w). (k) molar extinction coefficient: The molar extinction coefficient of the enzyme at 280 nm is about 1.08 x 105 M-Ú-cm-Ú. (1) Amino acid analysis: The enzyme is hydrolyzed with 6N-HC1 at 110 C for a predetermined time period (24-72 hrs.) and analyzed for amino acids by means of an amino acid analyzer. The values given in the following table denote the number of amino acid residues per molecule of the enzyme (mol. wt. = 4.8 x l0). EMI6.1 <tb> <SEP> Number <SEP> Number <SEP> Number <tb> Amino <SEP> acid <SEP> of <SEP> Amino <SEP> acid <SEP> of <SEP> Amino <SEP> acid <SEP> of <tb> <SEP> residues <SEP> residues <SEP> residues <tb> Lysine <SEP> 28.8 <SEP> Tryptophan <SEP> 14.4 <SEP> Serine <SEP> 20.2 <tb> Histidine <SEP> 13.9 <SEP> acid <SEP> <SEP> 47.0 <SEP> Aspartic <SEP> 47.0 <tb> Art <SEP> nine <SEP> 10.6 <SEP> Threonine <SEP> 18.7 <SEP> Proline <SEP> 13.9 <tb> Glycine <SEP> 23.5 <SEP> Valine <SEP> 22.6 <SEP> Leucine <SEP> 28.3 <tb> Alanine <SEP> 48.4 <SEP> Methionine <SEP> 5.3 <SEP> Tyrosine <SEP> 18.2 <tb> Cysteine- <SEP> 0 <SEP> Isoleucine <SEP> 10.6 <SEP> Phenyl- <SEP> 19.3 <tb> <SEP> alanine <tb> (m) Analysis for metal: As determined by the atomic absorption method, this enzyme contains about 1.5 gram-atom of manganese. It is, therefore, clear that the enzyme is a Mn-superoxide dismutase. The enzymatic potency assay procedure is as follows. The method described in the Journal of Biological Chemistry 244, 6049-6055 (1969) is employed. Thus, a sample cell (length of light pctth 1 cm) is filled with 1.5 ml of 100 mM phosphate buffer (pH 7.8), 0.5 ml of 60 M cytochrome C, 0.5 ml of 0.6 mM ethylenediaminetetracetate sodium, 0.25 ml of 0.6 mM xanthine and 0.21 ml of the enzyme solution diluted with phosphate buffer (pH 7.8) to a measurable concentration, followed by addition of 0.04 ml of xanthine oxidase solution to make a total of 3.0 ml. The cell is set on a spectrophotometer and the reaction is initiated at 250C so as to measure the initial velocity (v) of reduction of cytochrome C at 550 nm. As a blank, the. initial velocity (V) of reduction of cytochrome C in the presence of distilled water in lieu of the enzyme solution is also measured. With the amount of the enzyme causing a 50% inhibition of the initial velocity of reduction of cytochrome C under the above reaction conditions being taken as unity, the enzymatic potency is calculated by means of the following equation. = (v) x amount of (V) - (v) U/mg (v) x of or enzyme (mg)/l.0 ml reaction mixture Q The active dye method described in Analytical Biochemistry 44, 276-287, 1971 can also be utilized. Thus, after polyacrylamide gel electrophoresis, the gel is immersed in 2.45 mM nitro blue tetrazolium for 20 minutes. Then, the gel is further immersed in a mixture of 28 mM tetraethylethylenediamine, 0.028 mM riboflavine and 36 mM potassium phosphate (pH 7.8) for 15 minutes. The gel is put in a test tube and exposed to the light rays of a 15watt fluorescent lamp, whereupon the superoxide ion 2 generated by photoredue.e-ion of riboflavine reduces the nitro blue tetrazolium rj that the moiety other than that having superoxide dismutase activity forms a blue-formazan and is therefore dyed blue-purplish. Proteins can be assayed by the method of Lowry [The Journal of Biological Chemistry 193, 265-275, 1951]. The superoxide dismutase according to this invention differs in physicochemical properties from the known superoxide dismutase. The known superoxide dismutase includes the one obtained from Escherichia coli B [The Journal of Biological Chemistry vol. 245, No. 22, pp. 6176-6181, 1970] and the one derived from Bacillus stearothermophilus [Journal of Molecular Biology, vol. 105, pp. 333-335, 1976]. The molecular weight of the superoxide dismutase derived from Escherichia coli is 39,500 and that of the superoxide dismutase from Bacillus stearothermophilus is 40,000. Since these weight figures are significantly different from the molecular weight of the enzyme of this invention which is about 4.8 x 10 , the superoxide dismutase according to this invention is considered to be a novel enzyme. The superoxide dismutase of the present invention shows an overt inhibition of edema when a sample of 3000 U/mg is administered intravenously into the hind leg of the rat according to the carrageenan-induced foot edema method [Van Arman. C. G. et al: The Journal of Pharmacology and Experimental Therapeutics vol. 150, p. 328 (1965)] at the dose of at least 1.0 mg/kg. Effect to nystatin edema: To a paw of JCL-ICR mouse is subcutaneously injected 0.025 ml of 1% nystatin solution in physiological saline. After six hours from said administration, edema is emerged, and then superoxide dismutase (SOD) (11,000 U/mg.protein) of the present invention is subcutaneously injected to the portion. This experiment is conducted according to the method described in Pharmacology vol. 5, pp. 215-224(1971). The results are set forth in the following table. Dose doss No. of Edema volume(mg) Inhibition U/paw mice Mean + S.E. (%) Control --- 10 120.45 t 8.87 SOD 50 10 92.35 + 4.38 23.3 SOD 500 10 83.71 + 3.28 30.5 S.E. = standard error As is clear from said results, superoxide dismutase of the present invention shows a tendency of inhibiting nystatin edema. -The enzyme of this invention is sparingly toxic and no death is encountered even when it is intraperitoneally administered to rats at a dose of 60 mg/kg. Having an antiinflammatory activity, the superoxide dismutase according to this invention is of use as an antiinflammatory agent. For the treatment of various acute or subacute inflammatory diseases or edema (e.g. arthritis, bronchitis, edema due to a bruise, burn, etc.) in mammalian animals (e.g. mouse, rat, rabbit, cat, dog, monkey, man), the enzyme according to this invention can be administered orally or otherwise as formulated into tablets, capsule, injections, ointments or other known dosage forms at a daily dose of 1 to 5 mg/kg. Examples of the pharmaceutically acceptable carrier or excipient are albumin, globulin, dextran, Ficoll Type 70 (Sigma Co., U.S.A.), lactose, dextrin and starch. The following examples are given to illustrate this invention in further detail without limiting its scope. Unless otherwise specified, percents (%) are weight/volume percents (w/v %). Example 1 A 260 ml-conical flask was filled with 40 ml of a liquid medium containing 1.0% of solvent-extracted soybean neal, 1.5% of casein, 0 S% of diammonium phosphate, 0.1% of sodium chloride, 0.05% of potassium chloride, 0.02% of calcium chloride, 0.02% of magnesium sulfate, 1.0% of calcium carbonate, 0.0021% of zinc carbonate, 0.6% of soybean oil and 0.1% of Actocol (adjusted to pH 7.0), and was sterilized with steam at 1200C for 20 minutes. The flask was inoculated with a loopful of one of the undermentioned strain of the genus Serratia and incubated at 280C on a rotary shaker revolving at 230 r.p.m. for 48 hours. The fermentation broth was centrifuged to separate the cells which were lyophilized at -200C. A 10-gram portion of the cellular lyophilizate was taken and reconstituted at 300C. Following addition of 40 ml of 20 mM phosphate buffer (pH 7.8), the cells were disrupted by ultrasonication (2A, 5 minutes). The disrupted cells were centrifuged to give a supernatant fluid. This supernatant was measured for superoxide dismutase activity by the above-described xanthine-xanthine oxidase-cytochrome C method and polyacrylamide gel electrophoresis method. The results are set forth below in the table. It is clear that in all strains of Serratia, strong superoxide dismutase was intracellularly accumulated. Strain Deposit No. (I Activity (U/mg.protein) Serratia liquefaciens IFO 12979 74 Serratia marcescens IFO 3046 67 Serratia marcescens IFO 3759 115 Serratia marcescens IFO 3736 91 Serratia marinorubra IFO 12973 200 Example 2 (a) Harvesting the cells A 2-liter Sakaguchi flask was filled with 500 ml of a liquid medium of the same composition as described in Example 1 and, after sterilization, inoculated with Serratia marcescens IFO 3736. The flask was incubated at 280C on a rotary shaker revolving at 230 r.p.m. for 24 hours to give a seed culture. A 200-liter tank was charged with 100 Q of a medium of the same composition as above and under sparging at the rate of 100 Q/min., submerged aerobic culture was conducted at 280C. The resulting fermentation broth was centrifuged to separate the cells which were lyophilized at -200C. (b) Preparing a crude enzyme product A 1.5-kg portion of the wetted cell was reconstituted at 300C and after addition of 3 Q of 20 mM phosphate buffer (pH 7.8), the cells were disrupted in a Dynomill (Willy A. Bachofen Manufacturing Engineers, Switzerland) apparatus and centrifuged in a Sharples type centrifuge to give a clear supernatant. From this supernatant, the precipitate obtained with 30 - 708 (W/V) of ammonium sulfate was collected by centrifugation. -This sediment was dissolved in 20 mM phosphate buffer containing a small amount of 0.1 M KC1 (pH 7.0) and, in a cellulose tube, dialyzed against the same type of buffer for 3 days. The internal fluid, i.e. 150 ml of dialysate, was concentrated to 30 ml by ultrafiltration through a Dial low UM-5 membrane [an ultrafiltration membrane of Amicon Far East Limited, U.S.A.]. The concentrate was loaded onto a column (4.0 x 66.0 cm) of Sephadex G-150 previously equilibrated with the same buffer solution and eluted with the same buffer to collect 210 ml of an enzymatically active fraction. To this fraction was added ammonium sulfate to a concentration of 70% (W/V) and the resultant precipitate was collected by centrifugation and dissolved in a small amount of water. In a cellulose tube, this solution was dialyzed against water for 3 days and the internal fluid was lyophilized to give 620 mg of a crude enzyme product. The specific activity of this product was 3600 U/mg. protein. (c) Preparing a pure enzyme product (i) Column chromatography on diethylaminoethyl-cellulose In a small amount of 20 mM tris-hydrochloric acid buffer (pH 9.0) was dir;solved 620 mg of the crude enzyme obtained in (b), and in a cellulose tube, the solution was dialyzed against the same buffer for 3 days. The internal fluid, i.e. 80 ml of dialysate, was put on a column (2.5 x 45.0 cm) of diethylaminoethyl-cellulose previously equilibrated with the same buffer and the enzyme was eluted by increasing the concentration of sodium chloride in the same buffer linearly from 0 to 250 mM to collect 45 ml of an enzymatically active eluate. (ii) Gel filtration on Sephadex G-100 The enzyme solution obtained in (i) (45 ml) was put in a cellulose tube and dialyzed against 20 mM phosphate buffer containing 0.1 M of KC1. The internal fluid (60 ml) was concentrated to 6.0 ml by ultrafiltration through a Dyaflow UM-5 membrane. This concentrate was put on a column (2.6 x 74.0 cm) of Sephadex G-100 equilibrated with the same buffer as above and the enzyme was eluted with the same buffer to collect an enzymatically active eluate which was red-purple in color. The fractions thus obtained are constant in specific activity, and polyacrylamide gel electrophoresis shows that it is a single component product. The specific activity of this Mn-superc.xIde dismutase (24 mg) was 12,000 U/mg.protein. Example 3 Serratia marcescens IFO 3759 was treated in the manner as Example 2 to give 1.5 kg of lyophilized cells and, then, 410 mg of crude enzyme therefrom. The specific activity of this enzyme product was 3630 U/mg.protein. This enzyme was further purified by the procedure as Example 2 to give a pure Mn-superoxide dismutase product. The specific activity of this pure product was 12,000 U/g.protein. Example 4 Serratia marinorubra IFO 12973 was treated in the same manner as Example 2 to give 1.5 kg of lyophilized cells and, thence, 830 mg of crude enzyme product. The specific activity of this product was 3,800 U/mg.protein. Example 5 (a) Harvesting the cells. A 2000 ml of Sakaguchi flask was filled with 500 ml of a liquid medium containing 1.0% of solvent-extracted soybean meal, 1.5% of casein, 0.6% of diammonium phosphate, 0.1% of sodium chloride, 0.05% of potassium chloride, 0.02% of calcium chloride, 0.02% of magnesium sulfate, 1.0% of calcium carbonate, 0.0021% of zinc carbonate, 0.6% of soybean oil and 0.1% of Actocol (adjusted to pH 7.0), and, after sterilization, inoculated with Serratia marcescens ATCC 21074. The flask was incubated at 280C on a rotary shaker revolving at 230 r.p.m. for 24 hours to give a seed culture. A 200-liter tank was charged with 100 Q of a medium of the same composition as above and under sparging at the rate of 100 Q/min., submerged aerobic culture was conducted at 280C. The resulting fermentation broth was centrifuged to separate the cells which were lyophilized at -200C. (b) Preparing a crude enzyme product A 5-kg portion of the wetted cell was reconstituted at 300C and after addition of 10 Q of 20 mM phosphate buffer (pH 7.8), the cells were disrupted in a Dynomill apparatus and centrifuged in a Sharples type centrifuge to give a clear supernatant. From this supernatant, the precipitate obtained with 30 - 70% (W/V) of ammonium sulfate was collected by centrifugation. This sediment was dissolved in 20 mM phosphate buffer containing a small amount of 0.1 M KC1 (pH 7.0) and, in a cellulose tube, dialyzed against the same type of buffer for 3 days. The internal fluid, i.e. 530 ml of dialysate, was concentrated to 40 ml by ultrafiltration through a Diaflow-UM-5 membrane. The concentrate was loaded onto a column (4.0 x 66.0 cm) of Sephadex G-150 previously equilibrated with the same buffer solution and eluted with the same buffer to collect 550 ml of an enzymatically active fraction. To this fraction was added ammonium sulfate to a concentration of 70% (W/V) and the resultant precipitate was collected by centrifugation and dissolved in a small amount of water. In a cellulose tube, this solution was dialyzed against water for 3 days and the internal fluid was lyophilized to give 2070 mg of a crude enzyme product. The specific activity of this product was 3600 U/mg. protein. (c) Preparing a pure enzyme product (i) Column chromatography on diethylaminoethyl-cellulose In a small amount of 20 mM tris-HCl buffer (pH 9.0) was dissolved 2070 mg of the crude enzyme obtained in (b), and in a cellulose tube, the solution was dialyzed against the same buffer for 3 days. The internal fluid, i.e. 134 ml of dialysate, was put on a column (3.8 x 55.0 cm) of diethylaminoethyl-cellulose previously equilibrated with the same buffer and the enzyme was eluted by increasing the concentration of sodium chloride in the same buffer linearly from 0 to 250 mM to collect 54 ml of an enzymatically active eluate. (ii) Gel permeation on Sephadex G-100 The enzyme solution obtained in (i) (54 ml) was put in a cellulose tube and dialyzed against 20 mM phosphate buffer containing 0.1 M of KC1. The internal fluid (73 ml) was concentrated to 2.0 ml by ultrafiltration through a Dyaflow UM-5 membrane. This concentrate was put on a column (2.6 x 74.0 cm) of Sephadex G-100 equilibrated with the same buffer as above and the enzyme was eluted with the same buffer to collect an enzymatically active eluate which was red-purple in color. The fractions thus obtained are constant in specific activity, and polyacrylamide gel electrophoresis shows that it is a single component product. The specific activity of this Mn-superoxide dismutase (2.07 mg) was 12,000 U/mg.protein. Example of the preparation (1) An enteric capsule is obtained by per se conventional manner employing the following ingredients: Superoxide dismutase of the present invention (3,600 U/mg) 10 mg Lactose 46 mg Corn starch 16 mg Crystalline cellulose 12 mg Cellulose glycollic acid 5 mg Sorbitol ¯ 10 mg 99 mg (per capsule) (2) An enteric tablet is obtained by per se conventional manner employing the following ingredients: Superoxide dismutase of the present invention (3,600 Umg) 10 mg Lactose 79 mg Corn starch 45.5 mg Magnesium stearate 0.5 mg 135 mg (per tablet) Said two capsules or tablets are usually administered for adult human after meal, and the administration is carried out three times a day.

Claims: What we claim is: 1. Superoxide dismutase having the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pH range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability: Stable at 300C for 60 minutes; Inactivated about 50% at 50-600C in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 + 2.0%; H 7.05 + 2.0%; N 14.80 + 2.0%; (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.), 375-380 nm (min.), about 610 nm (shoulder); (j) molar extinction coefficient: about 1.08 x 105 M-Ú.cm-Ú at 280 nm. 2. Method uL producing superoxide dismutase characterized by cultivating a superoxide dismutase-producing microorganism belonging to the genus Serratia in a culture medium to thereby cause the microorganism to elaborate and accumulate superoxide dismutase in said medium and harvesting said superoxide dismutase from the resulting culture broth. 3. A method as claimed in Claim 2, wherein the microorganism is Serratia liquefaciens, Serratia marcescens, or Serratia merinorubra. 4. A method as claimed in Claim 2, wherein the micro organism is Serratia liquefaciens IFO 12979, Serratia marcescens IFO 3046, Serratia marcescens IFO 3759, Serratia marcescens IFO 3736, Serratia marcescens ATCC 21074 or Serratia marinorubra IFO 12973. 5. An antiinflammatory agent which contains an effective amount of superoxide dismutase having the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pH range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability: Stable at 300C for 60 minutes; Inactivated about 50% at 50-600C in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 t 2.0%; H 7.05 + 2.0%; N 14.80 + 2.0%; (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.), 375-380 nm (min.), about 610 nm (shoulder); (j) Molecular extinction coefficient: approx. 1.08 x 5 M -1 at 280 nm. as an active ingredient in association with a pharmaceutically acceptable carrier or excipient therefor. 6. A superoxide dismutase, for use as an antiinflammatory agent, having the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pH range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability; Stable at 300C for 60 minutes; Inactivated about 50% at 50-600C in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 + 2.0 %; H 7.05 - 2.0 %; N 14.80 + 2.0%; (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.), 375-380 nm (min.), about 610 nm (shoulder); (j) molar extinction coefficient: about 1.08 x 105 M-Ú.cm-Ú at 280 nm. EMI19.1 What we claim is: 1. A method of producing superoxide dismutase, characterized by cultivating a superoxide dismutase-producing microorganism belonging to the genus Serratia in a culture medium to thereby cause the microorganism to elaborate and accumlate superoxide dismutase in said medium, and harvesting said superoxide dismutase from the resulting culture broth. 2. A method according to Claim 1 characterized in that the superoxide dismutase has the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pfl range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability: Stable at 300C for 60 minutes; Inactivated about 50% at 50-600C in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 i 2.0%; H 7.05 - 2.0%; N 14.80 - 2.0%; (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.), 375-380 nm (min.), about 610 nm (shoulder); (j) Molecular extinction coefficient: about 1.08 x 5 M1 .cm1 at 280 nm. 3. A method as claimed in Claim 1 or 2, wherein the microorganism is Serratia liguefaciens, Serratia marcescens, or Serratia merinorubra. 4. A method as claimed in Claim 1 or 2, wherein the microorganism is Serratia liquefaciens IFO 12979, Serratia marcescens IFO 3046, Serratia marcescens IFO 3759, Serratia mareeseebs IFO 3736, Serratia marcescens ATCC 21074 or Serratia marinorubra IFO 12973. 5. An antiinflammatory agent which contains an effective amount of superoxide dismutase having the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pH range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability: stable at 300C for 60 minutes; Inactivated about 50% at 50-6Co in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 - 2.0%; H 7.05 - 2.0%; N 14.80 - 2.0%; (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.), 375-380 nm (min.), about 610 nm (shoulder); (j) Molecular extinction coefficient: approx. 1.08 x 105 M l.cm 1 at 280 nm. as an active ingredient in association with a pharmaceutically acceptable carrier or excipient therefor. 6. A superoxide dismutase, for use as an antiinflammatory agent, having the following properties: (a) Activity: to dismutate a superoxide ion into a hydrogen peroxide molecule and an oxygen molecule; (b) Substrate specificity: to act on superoxide ion; (c) Optimum pH range: about 7 to 8; (d) Stable pH range: about 6 to 11; (e) Optimum functional temperature range: about 20 to 450C; (f) Thermostability: Stable at 30 C for 60 minutes; Inactivated about 50% at 50-60 C in 60 minutes; Inactivated about 100% at 800C in 5 minutes; (g) Molecular weight: about 4.8 x 104 (by gel filtration method); (h) Elemental analysis: C 48.90 + 2.0%; H 7.05 - 2.0%; N 14.80 - 2.0% (i) Absorptions in the visible region of the spectrum: 470-475 nm (max.) 375-380 nm (min.), about 610 nm (shoulder)); (j) molar extinction coefficient: about 1.08 x 105M cm lat 280 nm.

Inventors: KATSUMI, TOMODA, KAZUTAKA, MAEJIMA, KOUICHI, MIYATA
Applicants: TAKEDA CHEMICAL INDUSTRIES, LTD.
Application year: 1981
Main classification: C12N9
IPC codes: A61K38, C12R1, C12N9