JPS58105998A - Protein an-7b - Google Patents

Abstract

NEW MATERIAL:Protein AN-7B having following propertes: molecular weight, 13,000-12,500; isoelectric point, 3.6; positive to ninhydrin and biuret reactions, negative to anthrone and Bricks reactions; soluble in water, insolube in ethanol, acetone, butanol; aminoacid composition in %: Asp 12.2, Thr 10.5, Ser 7.3, Glu 6.6, Pro 4.1, Gly 8.0, Ala 10.8, Val 8.9, Met 0.0, Ile 1.8, Leu 6.5, Tyr 0.9, Phe 5.5, Lys 2.0, NH3 9.4, His 0.7 and Arg 4.4. USE:Anticancer agent, antibacterial agent. PREPARATION:A microorganism in Streptomyces, e.g., Streptomyces griseoincarnatus B19C AJ0424 (FERM-P6012) is cultured, preferably aerobically at 4-9pH and 25-38 deg.C.

Description

[Detailed description of the invention] This invention relates to a novel protein AN-7B.

This protein (hN-7B) is present in the culture solution of actinomycetes of the genus Strebmyces, has antitumor t11 properties, and can be used in veterinary medicine.

The method for producing AN-7B is to produce AN-7B of the genus Streptomyces such as Streptomyces griseoincarnatus.
The AN-7B produced and accumulated in the culture solution may be collected from the culture solution by culturing a microorganism that has the ability to produce AN-7B. A
Examples of microorganisms that have the ability to produce N-7B include Streptomyces griseoincarnatus (Streptomyces griseoincarnatus).
ptomyus griseoincarnatus)
B 1 9 CA J 9 4 2 4 (F
ERM-PfLO'l2).

These microorganisms may be used to culture AN-7B in a conventional manner using a conventional medium containing a carbon source, a nitrogen source, an inorganic ion, etc., which allow the production and accumulation of AN-7B in the culture solution. As a carbon source, glucose, F.11. .

Sugars such as lactose, maltose □, rhamnose, glycerol, xylose, lactose, sucrose, starch, alcohols such as ethanol and sorbitol, and organic acids such as acetic acid can be used.

In addition to ammonium ions, amino acids,
Proteins, yeast fJ extracts containing these, casein hydrolysates, etc. can be used. Inorganic ions such as potassium ions, phosphate ions, calcium ions, magnesium ions, copper ions, zinc ions, manganese ions, iron ions, etc. are added to the medium as necessary.

Cultivation is preferably carried out under aerobic conditions, and more preferable results can be obtained by culturing while controlling the pH within an appropriate range of pH 4 to 9. The culture temperature is preferably in the range of 25r to 38C.

Methods for separating and purifying AN-7B from the culture solution include conventional protein methods such as salting-out method, gel filtration method, and adsorption chromatography.
Any method that can be used to separate and purify peptides can be applied.

The AN-7B thus obtained has the following properties.

(SDS polyacrylamide gel electrophoresis) (b)
White amorphous powder.

(c) Isoelectric point: 3.6 (by isoelectric focusing method)
(d) Ninohydrin reaction and Biuret reaction: both positive.

(C) A/Thron reaction and Brix reaction: both negative.

(f) rfl solution PC: soluble in water, ethanol,
Insoluble in acetone, butanol and propatool.

(g) Ultraviolet absorption spectrum: As shown in Figure 1e.

(h) Infrared absorption spectrum: As shown in Figure 2.

(1) Amino acid composition Asp 12.2% Thr 10.5 Set 7.3 Glu 6.6Pro
4.1 Gly8,0 Ala 10. FI Cys but not measured Vat 8.9 Met 0011 %11e
1.8 Leu 6.5 Tyr 0.9 Phe 5.5 Ly@ 2.0 NH39,4 )lie O,8 Trp Value not measured Arg 4.4 (j) Isoelectric point: a, 6 (isoelectric point electrophoresis).

(k) Although Bacillus subtilis ATCC 6633 and Sarcina lutea ATCC 9341 have antibacterial activity, their antibacterial activity against Escherichia coli ATCC 10798, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus FDA 209P is weak.

(1) Strongly suppresses the proliferation of mouse leukemia cells L1211+.

The value of AN-7B can be determined by the following method. That is, for a single purified product, it is usually carried out by the Buret method, Lowry method, UV absorption θ:, etc., which can be used for protein quantification.

In addition, when coexisting with other white matter, 1 moss polymer-permeable large intestine mutant strain MP-2 (FERM-P 5432) (Agr
ic, Biol, Chem,, 43.

371 (1979)) to perform a bioassay using antibacterial activity against MP-2 as an index. That is, a medium (MR medium) consisting of 1.75% Bacto Antibiotic Medium 3 (Difco product) and 1.0% agar.
After heat sterilizing at 120C for 35 minutes, dispense 2 Ome into Petri dishes and leave to cool to prepare a plate medium.

On the other hand, pebtone o, s%, extract 0.5%, NaC10,
A medium consisting of 3% agar and 0.8% agar is heat sterilized for 12 hours for 15 minutes. After that, keep it in a constant temperature bath at 42C until the temperature of the culture medium is 4.
After 2 CIs, MP-2, which had been previously cultured at 37r for 17 hours, was added to 1 Me medium in such a way that 5 x 10' cells were present. 2 by pipette
Collect 1 ml and add 1 ml onto the surface of the previously prepared M and 1 medium, quickly spread evenly and solidify.

Dilute the test solution containing AN-7B to 0.06
Soak m/ into paper disc (Toyo Roshi) #. This paper disk was cultured for 17 hours on a Hif plate 37c, and the size of the inhibition produced by AN-7B was measured.

Therefore, the distance (hu) from one end of the paper disk to the end of MP-2 growth inhibition is defined as the antibacterial activity of the solution containing AN-7B [J, , /- or 1119 AN-7B].
The antibacterial activity provided by -7B is expressed in terms of U/my.

The solution showing antibacterial activity of IU/me contained A N-7B 11.6 p? measured by the biuret method. /
Contains me.

AN'-7B has the effect of inhibiting the proliferation of mouse leukemia cells Ll+llo and can be used as an anticancer agent.

AN-7BH Neocarzinoatatin has an antibacterial effect on Sarunna lutea, etc.
) and other antibacterial proteins. Differences from already known antibacterial and antitumor proteins are shown in Table 1.

Table 1 Known anti-11'lt Ib2 protein AN-7B
Carcinocidinl) Colored protein, water-insoluble Melanomycin2)
Colored protein, water-insoluble A-2803) Colored protein, Iyomycin complex') Colored protein, water-insoluble at pH 2-4 Pluralin5) Colored glycoprotein, molecules 13 to 60,000 Actinoxanthin6)
Molecule 1t3. oo.

Amino acid composition Neocarzinostatir+7) Molecule i10,
700 UV absorption, amino acid composition Macromycin8) Sporamycin9) does not contain Met and Arg as protein-constituting amino acids Molecule JJ2.00
0 horizontal 2° amino acid ekoI (is, Arg, Met.

Does not include Cya or Pro.

2) R, Sugawara + A, Matsunae
and T, 1 lata: J, Antibio
tics+ 5erA Il+(4)+ I:U:+(+956).

3) Y, Sekjzawa, S, Inouye an
d K, Kagino: J, Antibiotic
s+ 5erA 15(6)+236(+96
2).

4) S, N1tta+ H, Yamamoto, A.
Matsumae and T., Hata: J.
, Antibiotics+ 5erA 17 (3
).

104 (1964).

5) H, Ogdwara, K, Maed
a+ K, N1tta+ Y, Okami
:J, Antibiotics+ 5erA 19
(1)+1 (1966).

6) Khokhlov, A.S.e.
T, al: J, Antibiot
ics+22, 541 (Wataru 969).

B) Chimura+ H, M, l5hizuka+
S., Hori, K.

Kimura+ J, Iwanaga+ T, T
akeuchi and H.

Umezawa: J. Antibiotjcs
+21.44 (1968).

9) Umezawa H, Komiyama H,
Takeshima, J.

Example (11A fq-7n production side) 20 scraps of the following medium A were put into a 500 m/capacity shoulder flask and killed.
'J culture solution was obtained. On the other hand, of medium B; (00M/! na 1
was placed in a fermenter and sterilized. This is 1 persimmon tar +><
A nutrient solution of 3 tl me was inoculated and cultured under aerobic conditions F at 30 r for 50 hours.

The titer in the obtained culture solution (antibacterial activity against E. coli MP-2V measured by the method described above) was 60 U.
/mJ (AN-7B 3611?, /me). 1 ton of the culture solution obtained by straining in this manner was
The cells were removed by centrifugation at Orpm for 20 minutes, the pH of the resulting liquid was adjusted to 4.0 with citric acid, and ammonium sulfate was added to bring it to 8(2) saturation.

51Tt After standing still for 30 minutes, 110000rp, 2 (1
A precipitate fraction was obtained by centrifugation for a minute. This precipitated fraction was dissolved in 100 rnN of B20 and dialyzed at 5C for 24 hours against a 5t010-fold diluted Macluvain buffer (pH 6, 0). The dialyzed fluid was centrifuged at 8.011 Orpm for 10 minutes to obtain a supernatant. 240 ml of slightly anion-exchanged seqq-7, DE-5 was added to the mixture, and the mixture was stirred with a trowel for 30 minutes while cooling with water. This was filtered using Toyo Roshi Ban 2 to obtain a filtrate. The pH of the filtrate was adjusted to 6.0 using citric acid. This solution was dialyzed at 5C for 24 hours against a 10-fold diluted maclupate buffer with H and 1O. l
:Jl is a weak anion exchange cellulose DE-52 column (
1,6X 40cjt+, flow rate 2-15 minutes) to adsorb the active fraction.

Wash with 0.002 M phosphate buffer (pH 7.2),
Next one 0. Elution was with 0.002 M phosphate buffer containing IM NaCl. The active ingredients are collected and processed into "Bio (Bi).

-Gel) P 301 column (color b, 1.6
X 40cn+)? Gel filtration was performed from this. (Flow rate 1
After dialysis with 1 t of water at 5C for 48 hours, the active substance AN-78+5 was lyophilized to obtain a white powder of the active substance AN-78+5.

The activity was 1667 U/my. Active substance AN-7B
is l'Blo-Ge1 JP-30 column 1lff
It is single with ff Togura Soi, [Column 1.6X4'
Oα, (1,02 M linoic acid-o, 5 M NaCl (p
H, 7, 2') buffer], a single band was obtained by 5DS-polyacrylamide gel electrophoresis.

The property of +21 A N -7B molecule was determined to be 12.4 x 103 using a 1Biogel p-aoJ column. [Column 1.6X
2 tl cm1 flow rate 3+++/15m1n, 0.0
2M phosphoric acid-0,1M NaCl buffer (+) T(7
, 2)'] When neocarzinostatin and cytochrome C, both of which have known molecular weights, were examined as standard substances, each had a molecular weight of 1.
It was 0700.13000. In addition, the molecule 'il'+ was measured by SDS polyacrylamide gel electrodynamic method.
j! It was 12.5×103.

The color reaction was as follows.

Ninhydrin reaction - Buret reaction - Anthrone reaction - Brix (B11x) reaction - *A+i treatment 1001T in NllCl, 411. ￥
1111 water was decomposed to liberate hexosamine.

Further, the purple-taton absorption curve of the active substance AN-7H at pH 7.1+ is shown in FIG.

Amino acid composition (using Hitachi KLA5-B automatic amino acid analyzer) Asp 12.2% Thr ILl, 5 3er 7.3 Glu 6.6 Pro 4.1 cry s.

Ala 10.8% Cys f8: not measured Va delivery 8.9 Met　　　　　0.0 11e 1.8 Le’u 6.5 Tyr, 0.9 Phe 5.5 Lys 2. O NH,,9,4 His O, 7 Trp amount not measured Arg 4.4 The solubility of this substance in solvents was as follows.

0Soluble in water.

0 Insoluble in ethanol, acetone, butanol, and propatool.

The infrared absorption Z vector of AN-7B was as shown by m22It. (KBr) Isoelectric point: The isoelectric point measured by electrophoresis method 1 is 3.6
Met.

(As a control, Ferichi/, bovine albumin and β
- When we measured lactochlobli/, it was 4.4 respectively.
．． They were 4.7 and 5.3. )GllA N-
Physical activity of 7B (1) The antibacterial activity of the active substance AN-7B was measured as follows. Various strains of M and I were inoculated in advance into a liquid medium and cultured at 37C for 20 hours. M:l k:'i medium to which the active substance AN-7B was added at a certain concentration (Bacto Antibiotic Ink Medium-a manufactured by Difco)
1.7 seconds, pH 7.2, sterilization 120C, 10 minutes) 2
+++et: 0.05 m/each inoculated with culture solutions of various strains cultured in advance, and cultured at 37C for 20 hours. 660
The growth rate of the bacterial strain was determined by measuring the absorbance at nm, and the minimum growth inhibitory concentration (MIC,') was determined (Table 2).

Table 2 MI C(/J?/me) ATCC10798 Enoerichia coli MP-21042 Bacillus subtilis 3 3 15
ATCC6633 Monkey 7 Na Lutea 1 1
2ATCC9: (41/Needomonas aeruginosa)100)
100 ) 100 ATCC 10145 Staphylococcus aureus ) 100
20 10FDA209P (iD RPMI ta 40 powder medium (Dainippon Pharmaceutical product)) Dissolve 0.38 f in 1 liter of distilled water, add NaHCO,l to 0.1%, and filter using a Millipore filter. The medium containing 100 ml of sterile serum was aseptically dispensed into each multiwell (Falco 7 product) by 1 rye, and pre-cultured mouse leukemia cells L.
12. . was inoculated o and o s me.

To this, AN-7D isolated and purified by the method of Example 1.2 was dissolved in the above medium at a concentration of 0.3/l? /me, 0
．． 5Ity/ml! Or 11+? /me concentration was added and cultured at 37C using a carbon dioxide gas incubator (7%, CO2 concentration) at 5F] to count the cells under a vertical microscope and count the proliferating cells. I looked up the numbers.

The results are shown in Table 3.

Table 3 Drug concentration Lljlllll number of cells 0
100 100
1000.30 50.
0 850.50
2.511 0 2
01.00 1.25
0 [1(iii) BOF 105 mouse leukemia cells"1.2eo I
,/CRJ was intraperitoneally transplanted into a 5-week-old female mouse, and the following E
], AN-7B was continuously staged for 5 days. The results were as shown in Table 4.1.

No. 4 Table Throwing 1 (/, /^7/number of times) V Average survival rate (A) - (Control) 9.0 ± 0.7 1002
'5X5 16. S±1. ．． 7 177

[Brief explanation of drawings]

In FIG. 1, a shows the ultraviolet absorption spectrum of AN-78 at pH 7.0 in, and b shows the ultraviolet absorption spectrum of neocarzinostatin, which was measured once for comparison. FIG. 2 is an infrared absorption spectrum (KBr) of AN-7B. Patent applicant: Ajinomoto Co., Ltd. Procedures Assistant ■゛Yu December 18, 1981 Commissioner of the Patent Office Shima 1) Haruki Tono2, Name of the invention! White 11AN-7B 3. Relationship between the person making the amendment and the case Patent applicant address: 5-8-5, Kyobashi-chome, Chuo-ku, Tokyo Number of inventions to be increased by the amendment None 0, Subject of the amendment
drawing

Claims (1)

[Claims] Isoelectric point of a protein 'QAN-7B0 (SDS polyacrylamide gel electrophoresis method) ('l) having the following properties:
:(, 6 (by isoelectric focusing) (C) Ninhydri reaction and Biuret reaction: both positive. (d) Anthro/reaction and Brix (B+'+x
)? l: Both negative. (e) Soluble AL property: Soluble in water, insoluble in ethanol, acetone, butanol and propatool. (f) Ultraviolet absorption spectrum: as shown in FIG. (g) Infrared absorption spectrum: as shown in FIG. 13. (h) Amino acid composition
3Asp 12.2% Thr 10.5%Set
7.3Glu
6.6 Pro 4.1 Gly, 8. tl Ala 10.8 Cys Amount not measured Val 8.9 Met O,0 11e 1.8 Leu fi, 5Tyr
O,9 Phe 5.5 Lys 2. O NH, 9, 4 Hist), 7Trp
Love is unmeasured Arg 4.4