KR830002535B1 - Process for preparing new biologically active substance - Google Patents

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Description

Process for preparing new biologically active substance

1 is a UV spectrum of the material 41,200RP obtained according to Example 1 of the present invention.

2 is an infrared spectrum of the same material.

The present invention relates to a novel biological agent (hereinafter referred to as designation number 41,200RP) consisting of cells isolated from a novel microbial culture designated as Micrococcus sedogenes M78, a preparation method thereof, and a pharmaceutical composition containing the same. .

41,200RP, a substance according to the present invention, is an amorphous, water-soluble white powder separated from Micrococcus sedogenes M78 (NRRL B-3505) and containing carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, chlorine, sodium and calcium. . Elemental composition of this material (calculated for dry matter) is C = 45-47%, H = 7.1-7.6%, O = 35-38%, N = 4.0-5.7%, P = 0.9-1.2%, Cl = 0.1-0.4%, S = 0.5% or less, Na = 2.0-3.0% and Ca = 0.9-1.9%, in anhydrous amino acids 11-18 (of which 5.5-7.5% alanine), glucose 10-17%, amino 10-17% sugar and less than 5% nucleic acid. In addition, when measured by a tablet (tablet) made of a mixture with the infrared spectrum kBr shows a main absorption skin as shown in the table in Example 1, it has an immunostimulatory, alkali metal salt and alkaline earth metal salt. 41,200RP is generally separated by 2 ~ 6% water content.

41,200 RP of the material according to the present invention was obtained by treating cells isolated from the Micrococcus sedogenes M78 strain with lysozyme, precipitating impurities using calcium chloride, treating with phenol to remove proteins, and then classifying the product with molecular sieves.

As a manufacturing method of 41,200RP by one feature of this invention

(A) Aqueous cell suspension of Micrococcus sedogenes M78 strain (NRRLB-3505) was maintained at 37 ° C. (range of 30-40 ° C.) at pH 6.5 to 8 for lysozyme. Processing,

(B) 32,919 RP materials in the form of alkali metal salts or alkaline earth metal salts by separating crude products from the liquid phase of the above suspension in the form of salts and continuously purifying impurities using calcium chloride. After obtained.

(C) It is characterized by collecting 32,919 RP in solution, that is, salt, using phenol, separating the product from molecular sieve, collecting high molecular weight components, and separating 41,200 RP. The cells used for the preparation of the aqueous suspension are dried in advance, and preferably heat-treated for 30 to 60 minutes at a pH of about 3 (range of 2 to 4) and a temperature of 120 ° C (range of 110 to 130 ° C).

In general, lysozyme is used at a rate of 2 to 20 mg per 1 g of dry cells. When treating with lysozyme, it is recommended to keep the pH constant at about 7 for 2 hours while vigorously stirring.

The liquid phase of the resulting suspension is usually separated by centrifugation and the dissolved low molecular weight product is removed using dialysis or ultrafiltration, which is passed through a membrane with a suitable porosity.

The solution of crude material thus obtained is lyophilized and separated in the form of a salt. The product obtained in this preparation step is composed of protein, nucleic acid, amino sugar and less than 5% of polysaccharide, and its elemental composition is outlined as C = 41.5 ~ 44.9%, H = 5.6 ~ 5.9%, O = 29.4 ~ 32.2% , N = 11.2 to 12.6%, P = 3.2 to 4.4%, S = 0.1 to 0.5%, and Cl = 0.2 to 0.4, and the sulfuric ash content is 11.4 to 15.4%.

It is common to purify by adding a concentrated solution of calcium chloride to a crude material of 10-40g per liter of water, usually 20g, until the final concentration of calcium chloride is 5-50g per liter, usually 10g.

The resulting precipitate is separated by centrifugation, and the resulting solution is dialyzed (or ultrafiltered) and then treated with an alkaline type ion exchange resin with sulfonic acid groups. The material (32,919 RP) thus obtained is separated from the solution by lyophilization. Substance 32,919RP is composed of 24 to 33% of amioic acid, 26 to 33% of nucleic acid, 11 to 17.5% of amino sugar (measured by Elson Morgan method, the result is represented by glucosamine) and polysaccharide 2.8 to 4.3%. In terms of the composition, C = 44 to 48%, H = 5.2 to 6.7%, O = 29 to 33%, N = 10.0 to 11.6%, P = 2.3 to 3.7%, S = 0.5% or less, and N = 3.0 To 3.8% and Ca = 0.15 to 0.50%.

32,919 RP of material is purified again in the following manner. In other words, a solution having a concentration of 32,919 RP of 10 to 50 g / l, usually 25 g / l, was prepared at 65 ° C using a mixture of the same solution of phenol and water (preferably 10% (w / w) of water). Most proteins are separated by washing for 15 to 60 minutes, usually for 30 minutes. The cooled liquid phase is separated and washed with chlorinated hydrocarbon solvent, usually chloroform, to remove dissolved phenol, and then dialyzed in middle water to freeze-dry the liquid phase containing the active substance.

The product thus obtained is dissolved in water buffered to pH 7 using an alkali metal of acetic acid such as sodium acetate, and the solution is used for molecular sieves having high porosity (e.g., "Ultregel Ac A22" from the Industrie Biologique Francaise, the Soci`et 'Sepharose 4 B' or 'Sepharose CL 4B' manufactured by 'e Pharmacia was fractionated to collect high molecular weight (5 × 10 ^{5 to} 5 × 10 ⁶ ) components. The material 41,200 RP obtained here is separated from the solution by dialysis in defoliated water for a long time and then lyophilized.

If necessary, the material 41,200 RP may be converted into an alkali metal salt or an alkaline earth metal salt using a known method. It is evident that the Micrococcus sedogenes M78 strain, a microorganism with a culture that makes cells used to make 41,200 RP under appropriate conditions, is a novel species entering the genus Micrococcus.

It is isolated from the soil collected in Brazil using conventional microbial separation methods. Samples of this bacterium were deposited and deposited in the NRRLB-3505 on August 14, 1968, at the Department of Agriculture, the Northern Regional Research Laboratory (NRRL). NRRL is an authority authorized to distribute strains to legally qualified individuals with relevant knowledge. The characteristics of these microorganisms were measured according to various known identification methods and other known methods summarized in the "Microbiological Method Handbook" published by the Society of American Bacteriologists, published by McGraw-Hill Book Company in 1957. The genus was determined according to the identification method described in "Bergey's Determination Bacteriology Handbook" (The Williams and Wilkins Company, Baltimore Publishing, 7th edition (1957)). Morphological characteristics of strain M78 were reviewed for fixed cultures maintained at 26 ° C for 24 hours. These cultures consist of immobilized, non-acid resistant P. aureus cells (diameter: 1 μ-1.3 μ), with two to four cells clustered or 4-16 cells forming a short chain or 20- It is a heterogeneous form of 50 cells. The presence of Mr. Hall was not observed.

The appearance of the culture on nutrient agar has a smooth surface and some very rough surface, and it has a greasy, oily coating of colorimetric body with odorless and soft consistency. Cultures of nutrient agars in Petri dishes are in the form of circular cells with uniform edges and smooth surfaces, which are flat or slightly convex and somewhat opaque. In the nutrient glucose solution, the culture has a moderate turbidity, no pigment, no odor, and a flake precipitate. When grown on potatoes, the M78 strain produces an extremely gray, greasy, oily coating that neither blackens nor contains soluble pigments.

The results of examining the biochemical characteristics of the culture cultured at 26 ° C are as follows.

Respiratory Type: Complete Oxygen Bacteria

Reduction of nitrates to nitrites: positive

Color Source: Voice

Indole Generation: Negative

Generate H ₂ S: Negative

Gelatin Liquefaction: Voice over 21 days

Casein hydrolysis: positive

Methyl Red Test: Negative

Acetyl-methyl-carbinol production test (Voges-Proskauer reaction): negative

Culture on skim milk: no aggregation, no peptonation

Alkalization of medium at pH6.1-7.5 between 1 and 21 days

Developmental temperature:

4 ℃-no development 30 ℃-good

22 ℃-Good 37 ℃-Moderate

26 ° C-Extremely good 45 ° C-No development

Catalase: positive

Oxidase: negative

The production of acid (in oxygen and anaerobic) from carbohydrates and polyhydric alcohols as measured by the color change of phenol red:

Glucose: negative Mannitol: negative

Galactose: negative Glycerol: negative

Arabinose: Negative Starch Hydrolysis: Positive

Sucrose: negative Escurin medium: no blackening

Lactose: Voice

Culture on medium with increased NaCl concentration:

4% NaCl: Normal development

10% NaCl: No development

Use of urea as a nitrogen source: negative

Urease: negative

Chitin Hydrolysis: Negative

Culture on autotrophic medium [added (Na ₄ ) ₂ SO ₄ as nitrogen source]: no development. No nitrite formation from Na ₄ ⁺ .

Following the principle of the Pridham method [Journal of Bacteriology, Vol. 56, pp. 107-114 (1948)], strain M78 utilizes the following materials with moderate to good efficiencies as carbon sources. Starch, ethanol, sodium acetate, sodium propioate, succinic acid, malic acid, sodium citrate and sodium pyruvate, the following are not used, or even poorly developed.

The source of nitrogen used for the development of ribose, arabinose, glucose, lactose, maltose, sucrose, trehalose, glycerol, mannitol inositol, glutaric acid, sodium tartarate, galacturonic acid, and M78 strain was determined according to the predam method. The method was measured by using sodium citrate as a carbon source and using various nitrogen-containing compounds in the medium instead of NH ₄ H ₂ PO ₄ . Under these conditions, the following compounds are used with moderate to good efficiency.

NaNO ₃ , NaNO ₂ , NH ₄ H ₂ PO ₄ , D, L-asparagine succinimide, glycine, D, L-alanine, D, L-aspartic acid D, L (+)-glutamic acid, L (-) -Tyrosine, D, L-proline The following are not used.

Adenine, uracil, creatinine, D (+)-glucosamine, sarcosine, arginine, L (+)-methionine, betaine.

In the verification method in the earlier "Verzy's Crystal Bacteriology Handbook", the bacterial cells examined did not contain photosynthetic pigments, and these cells did not develop on autotrophic medium and were spherical in shape and short in chain form. Alternatively, strain M78 can be classified as Buchanan (Buchanan, 1917), based on the fact that it is separated into a heterogeneous form and does not have a parent body.

On the one hand, the bacterium M78 is spherical, gram-positive and completely oxygen-free, reduces nitrate to nitrite, does not ferment carbohydrates under anaerobic living conditions, and does not form holes. Because of these characteristics, they can be classified as a globular family (Pribram, 1929).

The family is divided into six genera, which are divided into two groups according to their respiration type. By making this distinction, bacteria M78 can be classified into group 1, which includes cocci, staphylococci, Gaffkya, and falcobacteria. The first group is divided into two subgroups, which are characterized by cell division. The bacterial cells under review do not appear systematically in the four- or eight-cell crowd, and because of this property, strain M78 can be classified as a genus of cocci or staphylococcus. In particular, strain M78 can be classified as cocci because it does not ferment glucose under anaerobic living conditions and is completely oxygenated. In order to determine the genus much more accurately, various tests have been conducted to determine Staphylococcus aureus 209P, Neisseria catarrhalis A 152 (IP) Streptococcus faecalis ATCC 8043, and Streptococcus faecalis ATCC. 9790), and as a result, several of these genera were excluded and some of them could be classified as family other than cocci.

Among the 16 species belonging to the bacterium, the most similar to the strain M78 is Micrococcus ariane, Micrococcus caseoliticus and Micrococcus colphogenes.

In particular, strain M78 differs from micrococcus berrian in that it does not produce acid from glucose, lactose, sucrose, glycerol or mannitol and does not acidify milk, and also does not liquefy gelatin and does not peptonize milk, glucose, lactose It differs from Micrococcus caseoliticus in that it does not produce acid from mannitol or glycerol. In addition, strain M78 differs from Micrococcus colphogenes because it cannot hydrolyze chitin and has urea negative.

In summary, this strain is a new species different from the species in the micrococcus.

The preparation of the cells used to prepare 41,200 RP is to incubate micrococcus sedogenes M78 strain in a suitable medium under suitable conditions, and then isolate the proliferated cells during the culture.

In oxygen culture, micrococcus sedogenes M78 bacteria can be cultured by the surface method or the dipping method, but it is a suitable method because of the convenience of the dipping method. Inoculation, fermentation, and other types of equipment used for this purpose may be those normally used in the fermentation industry.

Fermentation broth should contain anabolic carbon, nitrogen, phosphorus, sulfur and minerals and, if necessary, growth factors. These ingredients may be added in the form of distinct products or in complex mixtures of different biological products with different sources.

As an anabolic carbon source that can be used, carbohydrates such as dextrin and starch, other carbon-containing substances such as alcohol (ethanol) or organic acids such as lactic and citric acid are used. Some types of animal or vegetable oils, such as lard oil or soybean oil, may be used as a substitute for these carbon sources. Suitable sources of anabolic nitrogen are extremely diverse, although inorganic or organic ammonium salts and extremely simple chemicals such as amino acids of this kind may be used and complexes containing mainly nitrogen in the form of proteins, namely casein, lactalbumin, gluten and hydrolysates thereof. Decomposition products, soy flour, peanut powder, fish meal, meat juice, yeast juice, soluble tailings (drinking juice) and corn steep liquor from distillation of grain alcohol may be used.

Sulfur and phosphorus are usually used in the form of the complexes listed above, but may be used in the form of sulfates and phosphates.

Some inorganic components to be added include phosphates of alkali metals, phosphates of alkaline earth metals, calcium carbonate or magnesium carbonate which have a buffering or neutralizing effect. To maintain the ionic equilibrium necessary for the development of the Micrococcus sedogenes M78 strain, chlorides and sulfates of alkali metals, chlorides and sulfates of alkaline earth metals or salts of zinc, cobalt, iron, copper and manganese are used. The pH of the fermentation broth at the start of cultivation should be 6.0-7.8 and should be between 6.5-7.5. A proper temperature range of fermentation is 25-30 ° C., but a satisfactory production can be achieved with a temperature range of 20-35 ° C. The aeration range during fermentation is extremely wide. However, it is particularly appropriate to use 0.3-3 l air / medium l / min. Depending on the medium used, the maximum yield is obtained after 8-20 hours, usually 15 hours after incubation. The pH range of fermentation broth at the end of incubation is usually 7.3-8.8, preferably 8.0-8.4. It is common to acidify the medium to pH of about 3 by adding an inorganic acid such as hydrochloric acid and then isolate the micrococcus sedogenes M78 microorganism from the fermentation medium by filtration or centrifugation.

After the above separation process, the produced crude cells may be used or washed with lyophilization or alcohol or acetone to dehydrate them, and then dried under reduced pressure to make purified dry cells. In particular, the prepared cells are heat-treated at 120-130 ° C. for 30-60 minutes, and then used for the next step. The novel material 41,200RP according to the present invention has remarkable and useful biological properties.

When administered intravenously, intraperitoneally, subcutaneously or intranasally in rats, the substance according to the present invention is characterized by the presence of malignant bacteria and meninges such as Listeria monocytogenes, Staphylococcus otheus and Escherichia coli. Normally dosed effects on viruses such as myocarditis virus, rat hepatitis virus, flu virus (type A ₀ , A ₂ ), herpes viruses, and Arbovirus from Semliki-Forest Resistance is large. Conditions that increase this resistance persist for at least 96 hours after treatment. In parenteral administration, 41,200RP promotes phagocytosis of reticulum endothelial system and increases the number of antibody-producing lymphocytes in the spleen. It also has a facilitating effect on reactions involving sustained-type and sensitization and reactions involving the production of antibodies to particulate antigens.

In rats with tumor cells (sarcoma 180 cells), 41,200RP induces necrotic reactions, thereby culling tumors. Under certain conditions, the product according to the present invention enhances the cellular disruption of P lymphocytes in the spleen against metastatic tumor cells. In animals (rats), the effective dose is usually in the range of 0.03-3 mg / kg body weight, depending on the method of administration and the experimental system used, preferably 0.3-1 mg.

In general, once a period of time is effective enough for at least 48 hours. For intravenous administration, the 50% lethal dose (LD ₅₀ ) of 41,200RP is 23mg / kg body weight.

As shown in the following, proteins are shown according to the results of amino acid analysis, glucose is measured by anthrone method, phosphorus is ashed, molybdate method, nucleic acid content is measured by absorbance at 258 mm, and amino sugar is Elson-. It is measured by morganan method and expressed as glucosamine or by hydrolysis and separation using an automatic analyzer (Biotronik LC 6000E), followed by ninhydrin.

The present invention will be described in detail according to the embodiment.

Example 1

(A) Cell production

Peptone (1,200g), meat juice (600g), soybean oil (1200CC) and tap water (amount that can make 110L whole) into 170L fermentation tank, adjust pH to 6.90 by adding 10N NaOH solution and then 122 ℃ Sterilize the medium by introducing steam for 40 minutes. When cooled, the volume of fermentation broth is 120 L and the pH of the medium is 6.70 due to condensation of steam during sterilization. Inoculated with the Erlenmeyer flask by culturing the Micrococcus sedogenes M78 strain (200CC). After incubating the culture with sterile air, it is preferable to incubate the resulting culture after growth at 27 ° C. for 14 hours.

Culture production was carried out in an 800 L fermenter containing:

L-lactic acid (4 kg), soybean oil (2 L), ammonium sulfate (2.4 kg), sodium chloride (2 kg), 1 calcium phosphate (0.4 kg), magnesium sulfate 7H ₂ O (0.8 kg), copper sulfate 5H ₂ O (0.02 kg) , Zinc sulfate 7H ₂ O (0.012 kg), cobalt chloride 6H ₂ O (0.00 kg), tap water (about 370 L total).

10N NaOH solution (2,950cc) is added to adjust the pH of the medium to 7.10, and then sterilize the fermentation broth by introducing steam at 122 ° C. for 40 minutes. Since the steam is condensed during sterilization, when the fermentation broth is cooled, the volume becomes 400ℓ and the pH becomes 4.20. The pH is adjusted to 6.60 by adding 5N NaOH solution (2,100cc).

Inoculate using 40 liters of inoculum culture from a 170-volume fermenter. The culture is grown for 16 hours at 27 ° C. with sterile air (15 m 3 / hr) and rotating the stirrer at 205 rpm. At the end of the operation, the culture medium had a pH of 8.20 and a fermentation broth of 440 liters. The juice thus produced is cooled to + 4 ° C. and pH is adjusted to 3 by adding 5N HCl (4 L). The cells are separated by centrifugation at 4,000 rpm (2,900 g). The cells are taken up in ethanol (80 L) at -10 ° C, stirred for 15 minutes, centrifuged and dried at a temperature of 35 ° C under reduced pressure (5 mmHg) for 15 hours to obtain dry cells (1,500 g).

(B) 32,919 RP manufacturing

The cells (1 kg) made in (A) are suspended in sterile distilled water (40 L) contained in a jacketed stainless steel reactor, circulated with water to adjust the temperature to constant temperature and agitate a high speed stirrer. 10N NaOH solution is added to adjust the pH to 7.0.

Lysozyme (2 g) is added and stirred at 37 ° C. for 2 hours, with the addition of 5N NaOH solution to periodically adjust the pH to 7.0. The mixture is centrifuged (using a Sharps M16 Machine, continuously passed at 17,000 rpm at a flow rate of 30 l / hr). The supernatant (37 L) was ultrafiltered (using a UFP 20 device equipped with an Iris 3042 acrylic film) to recycle the remaining material and added demineralized water (3 × 40 L) cooled to + 4 ° C. Collect the stock residue (7 L) without small molecules (molecular weight 25,000 daltons). 5N NaOH solution was added to freeze-dry the solution whose pH was adjusted to 7 to obtain crude product (147 g).

This product (31 g) was added to demineralized water (1 '550 cc) at about 20 ° C, stirred for 30 minutes to dissolve, and a solution containing 668 g / L calcium chloride hydrate (31 cc) was added over 5 minutes, with stirring being constant. Hold for 30 minutes. The resulting precipitate is separated by centrifugation (using a Sharps laboratory machine) at a flow rate of 5 L / hr at 40,000 rpm. The supernatant was dialyzed in deionized water (3 × 40 L) at a temperature of + 4 ° C. for 3 days using a cellulose membrane. The residue is stirred for 1 hour in a round bottom flask with sodium Dowex 50 × 2 polystyrene-sulfonic acid resin (310 cc) with stirring. The resin is filtered and washed on the filter with water (310 cc). The wash is mixed with the filtrate to lyophilize the whole. The characteristics of 32,919 RP (17 g) in the salt form thus obtained are as follows.

○ Appearance: White powder

○ Composition: Moisture (Fischer) = 15.7%

Protein (dry total amino acid) = 24.6%, polysaccharide = 3.9%, hexane = 28.9% for dry matter (by UV spectrum).

○ Atomic analysis: C = 46.30%, H = 5.84%, O = 30.71%, N = 10.04%, P = 3,11%, S = 0.5% or less, Na = 3.70%, Ca = 0.30%

32,919 RP tablets

32,919 RP (25 g) made under the conditions of Example (B) was dissolved in deionized water (1 L) in a 2 L volume of reaction weight with a stirrer and temperature controller, and a mixture of phenol and water (109 cc of water, 1 kg of phenol) (1 L ) Is heated at 65 ° C. for 30 minutes with vigorous stirring.

After cooling the reactor temperature to about 25 ℃ by using ice bath, each phase (phase) is separated for 30 minutes using a cooled centrifuge (Jouan, type K63F, capacity 4ℓ). Get 45cc The phenol phase and the intermediate phase were re-extracted with demineralized water (1 L) for 30 minutes at 65 ° C., cooled and centrifuged to obtain 1470 cc of liquid substance again. These liquid substances are mixed. The phenol is removed by continuous washing with chloroform (2 × 2 L). Centrifuge the liquid material at 3,300g for 30 minutes to obtain 1800cc liquid material. The clarified liquid is dialyzed against demineralized water (3 × 40 L) at 4 ° C. for 3 days. Lyophilized freeze-dried to give a white amorphous powder (14.5g).

This powder (8 g) is dissolved in 0.1 M sodium acetate sterile buffer (400 cc) at pH 7. [This buffer was prepared by dissolving analytical sodium acetate (136.09 g) in demineralized water (0.9 L) and adding acetic acid (d = 1.049) to adjust the pH to 7.0 before making the exclusive volume 1 L. Sterilize by heating at 122 ℃ for 45 minutes and sterilized using aseptic demineralized water at a rate of 1/10.

The prepared solution was placed in a cold room (+ 4 ° C) and poured into a column (Sepharose CL4B, diameter: 13.5 cm, height: 65 cm) treated with pH 7.0 using sterile 0.1 M sodium acetate buffer and the same buffer was added. Eluate at 1.33 L / hr to record the absorbance at 230 nm passing through 0.1 cm of the eluent. 3.03 liters of component is initially collected and discarded.

The absorption peak of the next component (1.22 L) is compatible with that for 230 nm, which is introduced into a tube of NOJAX regenerated cellulose and dialyzed for 3 days at + 4 ° C for demineralized water (3 × 40 L). The residue (1,300 cc) is lyophilized. The solution prepared by taking solids in demineralized water (93 cc) is dialyzed for 48 hours at + 4 ° C. against demineralized water (2 × 20 L) using the same membrane as above and the residue is lyophilized. This gives 41,200 RP (1.3 g) of material.

○ Appearance: White amorphous powder

○ UV spectrum: as illustrated in FIG.

○ Infrared spectrum (KBr disk): was about to receive the same as illustrated in FIG. 2 micron wavelength (top of the figure) and wave number cm ^-1 is the unit (bottom of figure) and vertical transmittance (%) Shown in the coordinates.

In the following table, the infrared main absorption band of the material 41,200 RP is represented by the wave number (cm ^-1 ).

Composition: Moisture (Ficher): 3.8%

About dry matter:

12.3% amino acids (7% of which are alanine)

Glucose 11.95% (below 1.3% of hexane)

16.3% amino sugar

Elemental analysis: C = 45.58%, H = 7.47%, N = 4.85%, O = 37%, P = 1.17%,

Cl = 0.25%, Na = 2.36%, Ca = 1.33%, S = 0.5% or less.

Electrophoresis: In a 1% W / V agarose gel with pH8.6 barbital buffer added and a voltage of 6 Vkm, 41,200 RP moves towards the anode at a rate of about 11 mm / hr [after oxidizing to periodic acid] Detecting products using either Schiff reagent or Sudan Black B].

Example 2

Follow the method of Example 1- (a). After 16 hours of incubation, the fermentation juice is cooled to 4 ° C. and the pH is adjusted to 3 by the addition of HCl. After separating the cells by centrifugation at 4000rpm, the acidic cell cake is salted for 45 minutes at 122 ℃ in the autoclave (autoclave). The cooled cells are washed with ethanol and dried. Subsequently, 41,200 RP (0.55 g) was obtained in the form of a white amorphous powder by carrying out the same amount according to the method of Examples 1- (b) and 1- (c).

O Composition: Water (Fischer): 6%

About dry matter:

17.7% amino acids (7.2% of which are alanine)

Glucose 10.5%

3% or less nucleic acid

Amino Sugar 15.%

O element analysis: C = 46.90%, H = 7.32%, N = 5.38%, O = 35%, P = 1.08%, Na = 2.26%, Ca = 1.83%, S = 0.5% or less.

The infrared spectrum of this product would be the same as that of 41,200 RP of material obtained in Example 1.

Claims (1)

(A) Treat the aqueous cell suspension of Micrococcus sedogenes M78 strain (NRRLB-3505) with lysozyme for 1-3 hours by maintaining the temperature at 30 ° -40 ° C. at pH6.5-8. ) The crude product is separated from the liquid phase of the above suspension in the form of salt, and purified by continuous precipitation of impurities using calcium chloride to obtain 32,919RP material in the form of alkali metal salt or alkaline earth metal salt, followed by (C) phenol. 32,919 RP in solution, that is, the high-molecular weight components are collected by treating salts and fractionating the product from the molecular sieve. 41,200 RP prepared by converting to a metal salt or alkaline earth metal salt, amorphous, water-soluble white powder containing carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, sodium and calcium. The composition (calculated for dry matter) is C = 45-47%, H = 7.1-7.6%, O = 35-8%, N = 4.0-5.7%, P = 0.9-1.2%, Cl = 0.1-0.4 %, S = 0.5% or less, Na = 2.0-3.2%, and Ca = 0.9-1.9%, in anhydrous state, 11-18% amino acid (4.4-7.5% alanine), glucose 10-17%, amino Infrared spectrum measured by a disk consisting of 10-17% sugar and 5% or less of nucleic acid and a mixture with KBr has a main absorption band as follows.

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