CA1209941A - Proteinaceous substance kud-pc and its production - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Disclosed herein is a novel proteinaceous substance designated as KUD-PC which is produced by culturing a KUD-PC-producing microorganism belonging to the genus Streptosporangium in a nutrient medium, accumulating the KUD-PC
in a cultured mass, and isolating said KUD-PC. KUD-PC is useful as an anti-tumor agent.

Description

~29~9941 NOVEL PROTEINACEOUS SUBSTANCE KUD-PC
AND ITS PRO~UCTION
This invention relates to a novel proteinaceous substance designated as KUD-PC and its production. More particularly, the present invention pertains to the novel proteinaceous substance KUD-PC
which is produced by culturing a proteinaceous substance XUD-PC producing microorganism belonging to the genus Streptosporangium and increases the antitumor activity.
We had found that a microorganism strain PO-3~7 belonging to the genus Strépto5porangium, isolated from a soil sample collected at Setagaya-ku, Tokyo, Japan, produced ~he antibiotic PO-357 (which has later,been designated as Sporamycin) having antibacterial activity against Staphy~ococcus aureus and Bacillus subtilis and antitumor activity. The physico-chemical properties of the said substance and taxonomical properties of the producing microorganisms strain were disclosed in Japanese Unexamined Patent Publication No. 53-7601.
We have found that a physiologically active substance, having no antibacterial activity, being electrophoretically differentiated from sporamycin and increasing the antitumor activity of antitumor substances, and have designated the said physiologically active substance as the protein KUD-PC.

12~9941 In the drawings attached to this specification:
~ Fig. 1 represents the W spectrum of KUD-PC;
Fig. 2 represents the IR spectrum of KUD-PC;
Fig. 3 represents an electrophoretic profile of KUD-PC analyzed by SDS-polyacrylamide gel electrophoresis;
Fig. 4 represents the electrophoretic pattern of cellulose acetate membrane electrophoresis of KUD-PC; and Fig. 5 represents the chromato~raphic pattern of KUD-PC, using a 'Biogel p_301TM column.
The novel protein KUD-PC of the present invention ~hereinafter designated as KUD-PC) has the following physicochemical properties.
~1) Elementary analysis: comprising carbon, hydrogen, nitrogen, oxygen and sulfur, - la -, ~

~2~9941 C 49.98%, H 7.27%, N 15.23~, S 1.08%, (found a~ter drying at 100C for 3 hours in vacuo), (2~ Molecular weight: 11500 calculated by comparison with standarcl substances (standard polypeptide or standard protein of molecular weight 2512 - 16949) by SDS-polyacrylamide gel electrophoresis, (3) Melting point: 258 - 260C (decomp.~, (4) Specific rotation: [~]D = -55.8 (c ~ 1.0, H O) (S) Ultra~iolet absorption spectrum: the UV spectrum of KUD-PC
in aqueous solution and 0~1 N-NaOH is shown in Fig. 1.
: 253, 259, 265, 268 tshoulder), 275, 280 (shoulder) nm, ~0 lN NaOH = 294 nm, (6~ Infrared absorption spectrum: Fig. 2 (KBr), (7) Solubility: soluble in water, and insoluble in common organic solvents such as alcohol; acetone and benzene, ~8) Color reactiono positive; Folin-Lowry, biuret and Rydon-Smith reactions, negative; phenol-~2SO4 and anthron-H2SO4 reactions, positive for ninhydrin reaction: HCl-hydro-~ysate, (~) Color and crystalline form: colorless crystals, crystals obtained by ultra-filtration of aqueous solution is hexagonal system crystals : - 2 -, .

iLZ~994:~

and by ammonium sulfate salting-out is prismic, (10) Nature: pH 4 - 8 ~0.1% aqueous solution of KUD-PC), 11) Amino acid analysis: KUD-PC (5 mg) is hydrolysed by HCl in a sealed tube and analysed by iiquid chromatography (Hitachi Model 034-2U), amino acid (~
aspartic acid 0.~ 4 4 threonine 0.2 5 8 serine Q 2 2 6 glutamic acid 0.1 4 1 proline 0.1 1 glycine 0~2 5 0 alanine 0.3 6 3 valine 0.2 6 5 isoleucine 0.0 1 6 leucine Q 1 1 7 methionine ~
tyrosine 0.0 2 0 phenylalanine - 0.0 7 9 lysine 0.~ 8 9 arginine Q D 4 0 histidine (trace) lZC~394~

(12) Electrophoresis: Electrophoretic profile analysed by SDS-polyacryla~ide gel electrophoresis - and drawn by chromato--scanner (5himazu CS-910) is shown in Fig. 3, as a single substance.
The electrophoretic pattern by cellulose-acetate membrane electrophoresis (Cellulo gel RS, pH 2, 1 M acetate-formate buffer, 200 V, 1 hour, amido black lOB
staining) is shown in Fig. 4, as a single band.
An object of the present invention is to provide a novel proteinaceous substance designated as KUD-PC having the physico-chemical properties hereinabove.
Another object of the present invention is to provide a process for the production of the no~el proteinaceous substance KUD-PC which comprises culturing the proteinaceous substance KUD-PC producing microorganism belonging to the genus-Streptosporan~_um, accumulating KUD-PC in a cultured mass and isolating the thus obtained KUD-PC therefrom.
The KUD-PC producing strain belongs to the genus Strepto~spor~ as described hereinabove, and the strain PO-357 belonging to genus Streptosporangium isolated by the present inventors are only illustrative for use effecti~ely in the present invention.
Taxonomical properties of the strain are as follows.

, , - .

~2~994~

1. Morphological properties.
Microscopic examination revealed that the aerial-mycelium is straight with most sporangia on the top of the mycelium. Size of sporangium is 5 ~ 10~, mean 7.5~ in diameter. Length of sporangia bearing aerial hyphae is short and mostly about 3~. Spores are round or ovoid with a smooth surface and a diameter of - 4a -~' 12~941 0O9-1~4~. Flagella are not observed.

2. Cultural characteristics on various media.
Observation at 27C, 10-14 days cultivationQ

Medium Growth Rever~e Aerial mycelium Soluble _ .
Sucrose-nitrate agar poor pale orange milk white none _ Glucose-a~paragine agar poor pale yellow milk white none _ _ _ _ Glycerol-asparagine ~gar poor pale yellow milk white none _ Inorganic salt-starch agar poor brownish milk white none . _ white-white _ .
Tyrosine agar moderate pale orange pale orange none _ _ ~utrient agar moderate orange orange none _ - good _ Yeast extract-salt extract agar good orange orange none _ . .
oatmeal agar good dark dark nsne _ orange orange _

3. Ph~iological properties.
51~ Growth temperature: possible at 43C, optimumat about 27-30C.
(2) Melanin production: negative on tyrosine agarO
(3) Liquefaction of gelatine: positive.

(4) Hydrolysis of starch: positive~

(5) Peptonization of milk: positive.

(6) H2S formation: negative.

(7) Nitrate reduction: positive.

a~94~;

4. Utilization of carbon sourceO
Observed on Pridham Gottlieb agar medium containing 0.05%
of yeast extract.
UtilizationO D-glucose, L-arabinose, D-xylose, D-mannose.
Probable utilization: D-galactose, D-raffinose, L-rhamnose, D-fructose.
No utilization: inositol, saccharose.
5. Cell wall composition.
Analysis of cell walls was made by the method of Becker et alO
[Appl. Microbiol., 13, 236-243 (1965)]o _ DAP* glycine arabinose galactose meso type _**

* diaminopimelic acid *-~ not detectedO
Various taxonomic properties hereinabove clearly reveal a strain PO-357 as a microorganism belonging to the genus Strepto-sporangium .

Among knownspecies, Streptosporangium pseudovulgalae Nonomura ~J. Ferm. Tech. Japan, 47, 701-709 (19Ç9)] is closely related to the strain PO-357, but there are differences, especially in the color of the aerial mycelium and in their biological activities.
Thus, strain PO-357 is designated as ~sg~g~E~s~y_em pseudo-vulgalae PO-357. The strain was deposited in the Institute for Microbial Industry, Agency of Industrial Sc ence and Technology and assigned deposit number FERM-P No. 3571.
In this invention, not only Streptosporangium sp. PO-357 but also the natural or artific$al mutants thereof, obtained by _ ~ _ 12~199~1 ultraviolet, X-ray, radiation or chemical mutagens, as well as the microorganisms belonging to the genus Streptosporangium having novel proteinaceous substance KUD-PC producing activity, can be utilized in the production of KUD-PC.
In the present invention, KUD-PC producing microorganisms belonging to the genus Streptosporangium are cultured in a conventional medium. The cultivation can be carried out in a conventional medium, in general, for ~treptomyces. As for the medium, a nutrient medium comprising assimilable carbon and nitrogen sources and inorganic salts can be used. As for assimilable carbon sources, glucose, sucrose, molasses, starch, dextrin, glycerin and organic acidsare used individually or in combination. As for nitrogen sources, peptone, meat extract, yeast extract, dry yeast, soy bean powder, corn steep liquor, cottonseed cake, caseine, soy bean protein hydrolysate, amino acids and urea are used indi~idually or in combination. If required, inorganic saltssuch as sodium, potassium, calcium, magnesium or phosphate salts are added. Further, trace nutrient elements or growth stimulants which accelerate growth of the microorganisms or production of KU~-PC can optionally be added.
Cultivation can preferably be perforrned under aero~ic conditions such as shaking culture or submeryed aeration culture at neutral pH at 27 - 30C
for 40 - 72 hours. KUD-PC is produced in a cultured broth. Cultivation should preferably be terminated at ~ , ' .

~2~994~

the highest accumulation of KUD-PC in a cultured broth.
Cultivation conditions such as culture composition, pH, temperature, agitation and aeration can be selected depending upon the strain used and other conditions.
Anti-foaming agents such as silicone oil, vegetable oil or - 7a -12~994~

surface active agents can be added if necessary.
Since KUD-PC imparted in the thus obtained cultured broth is mainly found in a cultured broth, KUD-PC can preferably be isolated fxom culture filtrate separated by filtration or centrifugation after optionally adding a filter aid. Isolation of KUD-PC
from the culture filtrate can be performed by applying the nature of KUD-PC in which the KUD-PC is soluble in water and insoluble in organic solvents such as alcohol, aceton~ or benzene. Generally, conventional isolation and purification methods for proteins or polypeptides can be applied. Salting-out by ammonium sulfate or ammonium chloride, fractional precipitation by methanol, ethanol or acetone, ion-exchange chromatography using an ion-exchanger, ion-exchange cellulose or ion-exchange 'Sephadex' , adsorption chromatography using active carbon, silica-gel, alumina, hydroxyapatite, cellulose or an adsorption resin such as HP-10* resin, gel-filtration chromatography using 'Sephadex'* or 'Biogel'**, electrophoresis, countercurrent distribution, dialysis, ultrafiltration or concentration procedure can be applied indi~idually or in combination thereof, or repeatedly. Elu~ion of chromatography can be carried out by water, aqueous alcohol,-aqueous acetone, buffer or aqueous solutions of inorganic or organic salts.
- An embodiment of the isolation and purification procedures for KUD-PC is illustrated in detail hereinbelow.

* Trademark ~or highly cross-linked ~ran containing functional ionic/grDups attached to gluoose units of the polysaccharide by ' ~ ether linkages.
~F~ ** Tra~Erk 12~99~

Cultured broth, added with filter aid, is filtered to remove mycelia. Ammonium sulfate is added to the culture filtrate at 90~ saturation under cooling at neutral pH. The salting-out precipitate was collected by filtration or centrifugation and dissolved in water or neutral buffer solution. The solution is dialysed under cooling or subjected to ultrafiltration using a - 8a -i -..
..

~2~999~1 -semipermeable membrane for desalting. An example of a semi-permeable membra,ne is a synthetic semi-permeable m~mbrane, which cuts off the molecular weightbelow 1,000 and over 10,000, such as UH-L*,, UK-10*
(product of Toyo Roshi Co., Tokyo).
De-salting solution is passed through a column of ion-exchange cellulose or ion-exchange 'Sephadex'TM after washing with water to elute by elution solvent.
Examples of ion-exchange cellulose are DEAE*-cellulose, ECTEOLA*-cellulose, SE*-cellulose and CM*-cellulose.
The above ion-exchange cellulose or ion-exchange 'Sephadex' is preferably bufferized with previously setting dilution concentration buffer solution. The elution liquid is a buffer,of an inorganic or organic salt. An example of an inorganic salt is sodium chloride, potassium chloride, ammonium chloride~
ammonium sulfate or a phosphate salt, and that of an organic salt is sodium acetate or sodium formate, with 0.01 - 1 M/1. An example of a buf~er solution is phosphate, acetate, citrate or trishydroxymethylamino methane buffer. The pH of ~he buffer solution is pH 5 - 8, preferably at neutral pH, at a concentration of 0.001 - 0.5 M/l. In the elution process hereinabove, concentrations of inorganic or organic salts and buffer solution,and pH, may optionally be changed gradiently or continuously.

g _ i' , * ~ra~rk lZ~99~

Concentration or cle-salting is performed by ammonium sulfate salting-out, ultrafiltration or dialysis.
Purification o:f concentrated or de-salted solution of KUD-PC is preferably performed by gel-filtration chromatography. An example of a gel-filtrating agent is 'Biogel P-10', 'Biogel P-30' or 'Sephadex G-75' (trademarks~. Elution is carried out with water or buffer solution.

- 9a -'i ~

12~994~ ' KUD-PC can be crystallized from the thus highly puri-fied solution of KUD-PC. For example, a highly purified eluate solution of KUD-PC by ion-exchange chromatography and/or gel-filtration chromatography is treated by ultrafiltration concentration and/or ammonium sulfate precipitation to obtain the crystalline KUD-PC. If not crystallized, ~UD-PC can be isolated by further chromatography or by a lyophilization pnx~dure.
Biological properties of KUD-PC are as follows.
1) Antimicrobial action:
KUD-PC does not show antibacterial action on test organisms such as Staphylococcus aureus, Bacillus subtilis and Bacillus cereus.
2) Cytoclasis action:
Cytoclasis (cytotoxic) action of KUD-PC is shown in Table 1, in which KUD-PC has strong cytoclasis activity on BHK-cells. Table 1.
KUD-PCsporamycin 3~-TdR 3H-UR Protein ~ e~5 U / me 42 ~ 2 2 I n 5 5 24 ~3 O 1~ . 55 30 42 0.1 ~S 27 ~13 22 2.5 2 6--2 7 2 8 I 0 2.5 4 2 0 1 7 û. l 5 4 91 4 ~ 2 3 ~--3 3 l) 1 0 5 3 3~1 2 2 3 Q l 1 0 8 ~4 3 5 2 1 0 6 8~1 4 4 1 ' 1 o 4 2 5 3 ~2~g9~1, i 3) Antitumor activity:
(1) Experimental method:
(i) Sarcoma 180, 1 X 105 cells, are inoculated subcuta-neousl~ in ddY mice. After 3 days mixture of sporamycin and KUD-PC is administered intraveneously. Further KUD-PC is administered intravenously at day 4, 5 and 6 to observe life-prolongation effects.
tii) Effect against Ehrlich ascites carcinoma:
Ehrlich ascites carcinoma, 2.5 X 106 cells, are inocu-lated intraperitoneally in ddy Mice (5 mice in one group, 5 weeks age). On next day, mixture of sporamycin and KUD-PC is administered intraperitoneally to observe life-prolongation days.
~2) Evaluation:
treated _ X 100 100 = life-prolongation ratio (~) (3) Combination effect of KUD-PC and sporamycin against animal transplantable carcinoma, Ehrlich ascited carcinoma is shown in Table 2-1 and -2, in which KUD-PC increases an antitumor acti~ity of sporamycin.
4) Toxicity:
Acute toxicity (LD50) of KUD-PC is as follows.
mice i.p. LD50 >2000 mg/kg mice i.v. LD50 >2000 mg/kg The follcwing examples illustrate the present invention but are not construed as limiting.
Example 1.
One loopful of microorganisms from a slant culture of Streptosporangium pseudovulgalae PO-357, FERM-P No. 3571 was inoculated into liquid medium A comprising glucose ~.0%, dry yeast 0.3~, peptone 0.5~, meat extract 0.5~, calcium .
~ ., -- 11 --~2(~94~

Table 2-1.

life-prolon- number of mice sample gation ratio ~60 days life-. (%)prolongation KUD ~P ~ sporamycir O~ o U/k~ O % 0/7 2.5 0 1 0 0/7 P . 13 0/7 0 3.000 35 n/7 2.5 3.000 ~5 2/7 1 0 3.000 70 3/7 0 6.000 43 1/7 2.5 . 6.000. ~11;1 ` ~/7 ~OQ0 >161 4/7 .

~, lZ~9~4~

Table 2-2.

-sporamycin ~ U D -P C number of 1 ~ 9~tLon ~ u ~ 3 ( ~1 itfie-prdolong- .~ . ~
_ .

2.5 2 9 3 8 . 0.~ 3 3 8 8 1 : . 0.1 5 3 2 5 2 _ _ 2 9 3 8 . 7 5 0 1 0 ~ 7 ~ 2 ~
. 2.5 3 3 5 7 . 0.6 3 4 5 1 1 ~
0.1 5 3 2 5 2 _ 2 6 .2 4 _ _ _ .
88 lo 23 lo 2.5 25 19 ~.6 3 4 3 1 05 L - 2 s L 1 9 ' ~ ~ .
;

~2~9~3141 carbonate 0.3% and sodium chloride 0.5%, (pH 7.0, 100 mi) in a 500 ml Sakaguchi flask, and shake cultured at 70 rpm at 27~ for 72 hours. This seed culture (10 ml~ was inoculated into medium B comprising glucose 0.2%, starch 1.5%, dry yeast 0.15%, peptone 0.23%, meat extract 0.3%
and calcium carbonate 0.25% (100 ml, pH 7.0) in ten 500 ~1 Sakaguchi flasks, and shake cultured at 170 rpm at 27C
for 48 hours. The cultured second seed (1 lit.) was inoculated into medium A tl30 lit.) in a 200 lit.
stainless steel tank, and submerged aeration cultured at aeration 100 lit./min., agitation ~50 rpm, inner pressure 0.5 kg/cm2, at 28C for 72 hours. KUD-PC was detected by cellulose acetate membrane electrophoresis in th~
cultured broth. The cultured broth (115 lit.) was cooled below 5C, added IHyflo Supercel' (trademark) (2.3 kg) and filtered by filter press. Filtered mycelia were washed with water, combined with the filtered broth, which was cooled below 5C, then added 'Hyflo Supercel'*
(400 g) therein, and added ammonium sulfate (82.5 kg) up to 90% ammonium sulfate saturation. The mixture was stirred at below 5C for 1 hour, and the thus obtained precipitate was collected to obtain wet solid material (900 g). The following operations were carried out in a dark roGm at below 5C.
The above solid material was dissoived in wa~er (3 lit.), filtered to remo~e insoiubie material and washed with water. The filtrate and washing solution were combined (total 4 lit.).

* Trademark for a ~ilter aid made of 'Celite' (trademark3 brand of dia~ceous e~h, ~ c ~ ly p ~ essed to provide a high filtrati~n rate.

12Q9g4~

~mmonium sulfate (2.5 kg) was added therets to prepare 90% saturation of ammonium suifate, stirred ~or 1 hour and the sedimentate was filtered to obtain the precipitated material (102 g), which was dissol~ed in water (450 mi).
The solution was dialysed with a ~Ceil~phane ? ** tube against de-ionized water (30 lit.) - 14a * *Trademark for a brand of regenerated ceilulose film.
produced from viscose by treatment with sulfuric acid and/or ammonium salts.

lZ~9941 for 3 days. De-ionized water was replaced ~nce every day.
The thus obtained de-salted soiutio~ ~1170 ml) was charge~ on a column (9 X 50 cm) of DEAE-cellulose (product of Brown Co., U.S.A., granule type), pre~iously bufferized with 0.002 M phosphate buffer (pH 7.5 - 8.0). Elution was carried out with 0.002 M phosphate buffer (pH 7.4, 6.4 lit.) and 0.01 M phosphate buffer (pH 7.4, 5 lit.), in this order, to fractionate each 30 ml fraction. Each fraction was checked by the Folin-Lawry protein detection method and bioassay using Staphylococcus aureus 209P, and found KUD-PC in fractions No. 181 -300 and sporamycin in fractions No. 321 - 400. Fractions containing KUD-PC
were collected (3.8 lit.) and ~mmonium sulfate was added therein to prepare a 40% saturation of ammonium sulfate, then the precipitate formed within one hour was filtered off. Ammonium sulfate was added to the filtrate to prepare 90% saturation, stirred for 1 hour and the precipitate collected, which was then dissolved in water.
The solution (78 ml) was charged on a column ~6.0 X 62 cm) of 'Biogel P-30' (trademark, Biorad Laboratories Co.), eluted with water for gel-filtration and each eluted fraction (20 g) was fractionated. Each fraction was checked by protein detection and bioassay as hereinabove explained to obtain fractions No. 41 - 54 (280 ml). The combined fractions were concentrated up to 10 ml by ultrafiltration membrane UH-l, and allowed to stand for overnight under cooling. The precipitated crystals were filtered, washed with a small amount of water and dried ~L2~994~

to obtain hexagonal system colorless cr~stals of KUD-PC
(120 mg). m.p. 258 - 260C (decomp.).
- The first mother liquor and washing solution were combined, ammonium sulfate up to 25~ saturation was added and the mixture - 15a -!, . ..~

lZ~ 41 allowed to stand for 3 days under coo~ing in a dark room.
The precipitated crystals were filtered, washed with a small amount of cold water and dried to obtain prismic colorless crystals of KUD-PC ~770 mg). The second mother liquor and washing solution were combined and ammonium sulfate was added therein up to 30% saturation to obtain crystals (560 mg). Further the third mother liquor was treated with ammonium sulfate up to ammonium suifate 60%
saturation to obtain crystals (450 mg). The thus obtained hexagonal system colorless crystals and prismic crystals were subjected to electrophoresis with a cellulose acetate membrance (CHEMETRON Co., 'Cellulogel RS'*, 5 X 12 cm) (lM acetate-formate buffer, pH 2, 200 V, 1 hour, stained by Amide Black 10 B). As shown in Fi~. 4, single identical bands were found in the same position.
The electrophoretic profile of SDS-polyacrylamide gel electrophoresis of hexagonal system colorless crystals is shown in Fig. 3 (scanned by scanner, Shimazu CS-910?, in which a single band is foundO
Example 2.
The main cultured broth (116 lit.) obtained by the same process as shown in example 1 was treated in the same manner as in example 1 to obtain a de-salted solution (1080 ml). The solution was charged on a column (9 X 50 cm) of DE~E-cellulose, pre~iously bufferized by 0.002 M phosphate buffer (pH 7.0 - 7 .4?, purified by the same process as in example 1 to obtain KVD-PC and sporamycin containing fractions No. 121 - 260.

* Trademark ,, ~ZC19941 The fractions were combined and treated in the same way as in example 1 for ammonium sulfate saturation 90% and 'Biogel P-30' column chromatography to obtain KUD-PC in the fraction No. 41 - 50 and sporamycin in the fraction No. 53 - 64. The 'Biogel P-30' coiumn chromatographic pattern is shown in Fig. 5. The fractions containing KUD-PC were treated by ultrafiltration and crystallization in the same way as in example 1 to obtain hexagonal system colorless crystals (730 mg) Im.p. 258 - 260C (decomp.)J , prismic crystals (1.48 g~
and cxystals (0.9 g)~ These crystals were found with a single band at the same electrophoretic position by cellulose acetate ~embrane electrophoresis.
Example 3.
The main cultured broth obtained by the same process as in example 1 was purified by the same way as in example 1 to obtain ammonium sulfate saturation 40 - 90% precipitate of fractions containing KUD-PC by DEAE-cellulose column chromatography. The precipitate was dissolved in water (70 ml), and charged on a column (6 X 78 cm) of 'Sephadex G-50l, previously swollen with water, for gel-filtration chromatography.
Eluate by water was fractionated each 20 g fraction.
Each fraction was checked by protein detection to obtain fractions No. 56 - 80 containing KUD-PC. The solution was de salted and concentrated by ultrafiltration~ and the concentrate (100 ml) was lyophilized to obtain white powder KUD-PC (1.45 g)~

`v The product showed a single band by cellulose acetate membrane electrophoresis and confirmed the same substance of hexagonal system colorless crystals and prismic crystals as was obtained in example 1.
Example 4.
As in example 1, fermentation in a 200 lit.
tank was carried out for 96 hours to obtain a main culture broth (114 lit.). KUD-PC was detected in the broth by cellulose acetate membrane electrophoresis and sporamycin was not found by bioassay.
The main culture broth was purified by the same process as in example 1 to obtain hexagonal system colorless crystals (50 mg~ Im.p. 258 - 260C (decomp.)]
and prismic crystals (1.89 g). These crystals were shown to have a single band by cellulose acetate electrophoresis.

Claims (12)

The embodiments of the invention in which an exclusive property of privilege is claimed are defined as follows:
(1) A process for the production of a novel proteinaceous substance designated as KUD-PC which has the following physico-chemical properties:

1) Elementary analysis: comprising carbon, hydrogen,nitrogen, oxygen and sulfur;
C 49.98%, H 7.27%, N 15.23%, S 1.08%,

2) Molecular weight: 11,500 (SDS-polyacrylamide gel electro-phoresis),

3) Melting point: 258 -260°C (decomp.),

4) Specific rotation: [.alpha.]D20 = -55.8° (c = 1.0, H2O),

5) Ultraviolet absorption spectrum. as shown in Fig. 1,

6) Infrared absorption spectrum: as shown in Fig. 2,

7) Solubility: soluble in water, insoluble in organic solvent (alcohol, acetone and benzene),

8) Color reaction: positive: Folin-Lowry, biuret and Rydon-Smith reactions, negative: phenol-H2SO4 and anthrone-H2SO4 reactions, HCl hydrolysate: positive for ninhydrin reaction,

9) Nature: pH 4 - 8 in 0.1% aqueous solution,

10) Color and crystalline form: colorless crystals,

11) Amino acid composition: Molar ratio of amino acid analysis of HC1 hydrolysate is as follows;

aspartic acid 0.1 4 4 threonine 0.2 5 8 serine 0.2 2 6 glutamic acid 0.1 4 1 proline 0.1 1 1 glycine 0.2 5 0 alanine 0.3 6 3 valine 0.2 6 5 isoleucine 0.0 1 6 leucine 0.1 1 7 methinine -tyrosine 0.0 2 0 phenylalanine 0.0 7 9 lysine 0.0 8 9 arginine 0.0 4 0 histidine (trace)

12. Electrophoresis: Electrophoretic pxofile of SDS-poly-acrylamide gel electrophoresis is shown in Fig. 3 and the substance shows a single peak; which process comprises culturing the proteinaceous substance KUD-PC
producing microorganism Streptosporangium pseudovulgalae PO-357 FERM-P No. 3571 in a nutrient medium, accumulating the protein-aceous substance KUD-PC in a cultured mass, and isolating the thus obtained protein-aceous substance KUD-PC therefrom.
(2) A novel proteinaceous substance designated as KUD-PC having the following physicochemical pxoperties:
1) Elementary analysis: comprising carbon, hydrogen, nitrogen, oxygen and sulfur;
C 49.98%, H 7.27%, N 15.23%, S 1.08%, 2) Molecular weight: 11,500 (SDS-polyacrylamide gel electro-phoresis), 3) Melting point: 258 -260°C (decomp.), 4) Specific rotation: [.alpha.]D20 = -55.8° (C - 1.0, H20), 5) Ultraviolet absorption spectrum: as shown in Fig. 1, 6) Infrared absorption spectrum: as shown in Fig. 2, 7) Solubility: soluble in water, insoluble in organic solvent (alcohol, acetone and benzene), 8) Color reaction: positive: Folin-Lowry, biuret and Rydon-Smith reactions, negative: phenol-H2SO4 and anthrone-H2SO4 reactions, HC1 hydrolysate: positive for ninhydrin reaction, 9) Nature: pH 4 - 8 in 0.1% aqueous solution, 10) Color and crystalline form: colorless crystals, 11) Amino acid composition: Molar ratio of amino acid analysis of HC1 hydrolysate is as follows;

aspartic acid 0.1 4 4 threonine 0.2 5 8 serine 0.2 2 6 glutamic acid 0.1 4 1 proline 0.1 1 1 g1ycine 0.2 5 0 alanine 0.3 6 3 valine 0.2 6 5 isoleucine 0.0 1 6 leucine 0 1 1 7 methinine tyrosine 0.0 2 0 phenylalanine 0.0 7 9 lysine 0.0 8 9 arginine 0.0 4 0 histidine (trace) 12) Electrophoresis: Electrophoretic profile of SDS-poly-acrylamide gel electrophoresis is shown in Fig. 3 and the substance shows a single peak, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.