JPS5819277B2 - Live Earth Transcriptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing Revistin, an inhibitor of cancer virus RNA-dependent DNA polymerase, which was discovered by the present inventors. The present invention relates to a method for producing lepistin obtained by culturing the E397-1 strain.

The present invention relates to a method for collecting and purifying this specific inhibitory substance, lepistin, and its use as a drug or reagent.

According to the invention, Levistin includes all those present as free acids, as their salts with various metals or organic bases, and in crude, purified and pure forms. .

The presence of an RNA-dependent DNA polymerase, ie, Liguarth transcriptase, in RNA tumor viruses is known.

(Tem1n, H-M, and Mizutani
, S,: RNA-dependent DNA
Poly-merase 1nVirions o
f R8V, Nature 226 el 211
(1970) s Baltimore D.

: Viral RNA dependent DN
A Polymerase -Na -ture
226, 1209 (1970)).

Therefore, the substance that inhibits the activity of the enzyme Vers transcriptase, which is specific to this RNA cancer virus, is (1
) It is expected to be used as a cancer preventive drug to prevent carcinogenesis, C) as a cancer treatment drug or an adjunct to cancer treatment, and (3) as a reagent for biochemical experiments.

The present inventors have developed a mouse leukemia virus called 6 Raus.
cher 1 eukemia V 1-rus (
In a therapeutic experiment on mice that had been infected with RI;V),
□I was able to recognize its effectiveness.

Resvitin is an active substance produced by the present inventors in the culture medium of microorganisms such as actinomycetes, molds, and mushrooms that has the effect of inhibiting nucleic acid synthesis by murine leukemia virus RNA transcriptase. Discovered through a systematic search.

Next, we will describe the bacteria that produce resvitin.

An example of resvitin-producing bacteria used in the present invention is
There is strain E397-1 (Feikoken Bacterial Serial No. 2869) belonging to the genus Streptomyces.

This E397-1 strain was isolated by the present inventors on April 3, 1971 from the soil of Oaza Ideinohatake, Oyama City, Tochigi Prefecture.

The mycological properties of this E397-1 strain are as follows.

(a) Spore chains are formed on the formed aerial hyphae, and each spore chain usually carries ten or more spores and exhibits a loose circular, hook-shaped, or spiral shape with three or more coils.

In electron microscopy, the spores are cylindrical (0.5-0.7X
1. O~2.4μ) with strains on the surface.

(b) Growth status on various media 1) Sucrose/nitrate agar (Waxman medium A 127°C
Culture): Good growth.

The back side is light-colored. Aerial mycelia are pearly powdery and form dewdrops.

Produces a buttery yellow, diffusive pigment.

This dye does not change with 0.05N caustic soda or 0.05 Num.

2) Glucose asparagine agar (Waxman medium A2
．． 27°C culture number): Good growth.

The back side is brown.

It forms light yellowish-gray to yellowish-gray aerial mycelium and produces a light yellowish and diffused pigment.

3) Glycerin-asparagine agar (ISPI nte
rnat'1onal S treptomyces'
Project) Medium A5', 27°C culture)
: Good prison education.

The nose is dark brown.

It forms a solid yellowish-gray powder and produces a dark-brown, diffused pigment.

Young cultures of 4 days old produce a yellow pigment, which turns brown with 0.05N caustic soda. (The original pt<< does not return with acid.

6'4) Ordinary agar (Waxman medium $4,
(Culture at 27°C): Colorless and grows considerably.

Does not form aerial mycelium.

35) Inorganic salt/starch agar SP medium'□A 4 which produces a pale yellow-brown, diffusive pigment.

27°C culture) Good growth.

The underside is yellowish brown to dark yellowish brown.

It forms yellow-gray to yellow-peach-gray powdery aerial mycelium, and produces a light wheat-colored diffuse pigment.

Hydrolyze starch. 6) Good growth on oatmeal agar (ISP medium A3, cultured at 27°C).

The back side is pale yellow. It forms yellow-gray powdery or velvety aerial mycelia and produces light yellow-brown diffuse pigment.

7) Yeast extract/malt extract agar (ISP medium A2
, 27°C culture): Very good growth The underside forms brown, yellowish-gray to yellowish-peach-gray velvety mycelia, producing a light brown, diffuse pigment.

After 4 days of culture, it rapidly produces a pigment that does not change color with acids or alkalis.

8) Synthetic nitrate Pros (cultured at 27°C): Produces a pale yellow soluble pigment, which changes to bright yellow with 0.05N caustic soda, and returns to normal color with acid.

9) Nutrient agar (Waxman medium A14, cultured at 27°C): Colorless and grows considerably.

Does not form aerial mycelium.

A pale yellow-brown diffuse pigment produced on Nishin Agar (IS)
P culture at 7, 27°C) Two good growth.

The underside is dark brown. It forms pearly, powdery aerial mycelium and produces a blackish-brown, diffused pigment.

(c) Physiological properties l) Growth temperature range: Glucose.

Basically, asparagine agar, yeast extract, and malt extract grow well to very well at 20°C, 27°C, and 37°C, but do not grow at 50°C.

2) Liquefaction of gelatin (on glucose/peptone gelatin medium) = Slight liquefaction is observed after 3 weeks.

3) Confirmation of starch hydrolysis (inorganic salt/starch agar medium): Positive, 4) Coagulation and peptonization of skim milk: Negative, weak peptonization is observed.

5) Production of melanin-like pigment (on tyrosine agar medium, glucose peptone gelatin medium, tryptone yeast pronu medium): Positive, but negative on peptone yeast and Toex iron agar medium (ISP medium A6).

6) Reduction of sulfate (nitrate broth and synthetic nitrate broth, Ross medium) double negative 7) Production of hydrogen sulfide (on protease peptone gucose agar): positive (detected with lead acetate paper).

8) Assimilation of carbon sources (on Puri de Mu Gontrieb agar base medium): (i) Use: D-glucose, L-arabino,
-su, Shukanu, D-xylose, i-inositol, D-mannitol, D-7 lactose, raffinose.

(i) Utilization 1jf=b: Comparing the above properties of Rhamnonu cellulose E-397-1 strain with those of known actinomycetes, Streptomycesfila
ipinensis ANVIONN, GOTTLIEB
.

BROCK, CARTERandWH, IT FIELD
1955: Streptomyces durha
menesis GORDONandLAPA 19
66: Streptomyces griseoc
hro-mogenecs FUKUNAGA, MI
SATO, l5HIIand ASAKAWA 195
5; Streptomycesalanosin
icus THIEMANN and BERETTA
Although it has common properties with the four types of actinomycetes identified in 1966, strain E397-1 differs from these four types of actinomycetes in the following points.

Streptomyces filipinensis
The 15P5112 strain forms pale brown-gray aerial mycelia on yeast extract/malt extract agar, oatmeal agar, inorganic salt-starch agar, and glucose/asparagine agar.

The pale yellowish brown color on the underside is much lighter than that of the E397-1 strain.

Various media, including synthetic nitrate prosthesis, produce no or only traces of a pale bluish-yellow pigment, which diffuses.

S treptomyces durhamensi
s I S P 5539 strain has aerial fungi, the color of threads, the color of the back side growing on the medium, and the diffusible pigments of S, fili.
They are almost the same as those of the S. pinensis I SP511 strain.

Coagulate milk. Streptomyces gri
secchromogenes l5P5499 strain,
The sharpness of aerial mycelium, the color of the back side, and the diffusible pigment in various media are S.
, filipinensis ISP 5112
strains and S. durhamensis I SP
It is very similar to those of the 5539 strain.

Reduces phytosate to nitrite.

Forms pale yellow-brown gray (pearl-colored) aerial mycelium on ordinary agar medium.

S treptomyces alancsinic
The s I S P 560 f5 strain forms a spiral with five or more turns on a glycerol-asparagine agar medium.

Forms light brown-pink aerial mycelium on oatmeal agar, inorganic salts, and starch agar medium.

In various media, it does not produce a diffusible pigment, or it produces a slight bluish-orange-yellow diffusive pigment.

Thus, strain E397-1 is Streptomyce
Although it is most closely related to sf 1lipinensis, it is distinguished from this in the following points.

That is, the production of a pH-sensitive pigment in synthetic nitrate prosthesis, which colors the back side of growth, and the production of a pigment that diffuses into the medium (the color of aerial mycelia).

Others include S treptomycesfiljpi
nis is an antifungal polyene antibiotic.
strain E397-1 produces antibacterial substances.

Although actinomycetes are prone to mutations both artificially and naturally, the present invention can use Streptomyces E397-1 strain and all of its mutants.

The revistin-producing bacteria referred to in the present invention include all strains belonging to the genus Streptomyces as long as they produce revistin.

Levistin can be obtained by inoculating spores or hyphae of a lepistin-producing bacterium, eg, Ebastreptomyces E397-1 strain, into a nutrient-containing medium and growing aerobically.

As the nutrient source, those known as nutrient sources for actinomycetes can be used.

For example, commercially available glycerin, glucose, lactose,
Carbohydrates such as sucrose, starch, maltose, molasses, or carbon sources such as fats and oils, and commercially available peptones, meat extracts, corn steep liquor, cottonseed oil, peanut flour, soybean flour, yeast extract, N-Z-7mine. Nitrogen sources such as , casein, sodium nitrate, and ammonia nitrate and inorganic salts such as common salt, phosphate, calcium carbonate, and magnesium sulfate can be used.

In addition, a trace amount of metal salt is added as necessary.

These substances may be used by levistin-producing bacteria and may be useful for the production of levistin.

All known culture materials for actinomycetes can be used.

Liquid culture is preferred for mass production.

The culture temperature may be within a range that allows lepistin-producing microorganisms to grow and produce levistin, but a temperature of 25 to 35°C is particularly preferred.

Ru.

Cultivation is usually continued until sufficient lepistin has accumulated.

For example, glycerin 2.5%, polypep): /1.0%
A medium prepared by dissolving 0.2% yeast extract and CaCO30.6 in tap water was adjusted to pH 6.8JC and sterilized, and then inoculated with spores and mycelia from a slope culture of Levistin-producing bacteria such as Streptomyces strain E397-1. When cultured with shaking aerobically at 27°C, accumulation of levistin was observed on the 3rd to 5th day of culture.

Lepistinase transcriptase (RN)
quantified by inhibition of A-dependent DNA polymerase).

As a quantitative method, the inhibitory activity was measured as follows.

Purified murine leukemia virus MLV was used as a source of enzyme transcriptionase.

The total number of reactions was 0.12rrLt, which contained the following components.

Trie (pH 8,1) 4.8 μmol, MgCl
20.3 mol, Na Cl 3.6 tt mole, dit
hiothreitolO, 012 mol, Non1de
t P -402,4xlO'm7*dATP0.01
2”Ele, dCTPO, 012ttmol, dGTPO,
o 12tt mol, 3fK m et ly1→TTP
6 μci, poly(dt)poly(A)
copolymer 3μg, MLV protein amount 2.16μg, test solution 0.025m, /:o reaction solution was incubated at 37°C for 60 minutes with shaking, then the o, 1r
rLt was applied to a circular filter paper with a diameter of 2.4 cm, which had been previously soaked in 0.1 M biophosphate solution and dried, and the filter paper was immediately shirred into an ice-cold 5-part solution.

Use 10 ml of cold 5STCA solution per filter paper.

After 1 minute, discard the liquid and wash the filter paper twice with cold 5STCA solution and once with cold 95% ethanol, then dry.

Place the filter paper in a vial and add toluene-ppo-p.
An opop scintillator was added, and the radioactivity of 3H=TMP incorporated into the acid-resistant fraction was measured by liquid entrapment, and the 3H-TMP value ('r) of the test group and that of the control group were compared. ), the RNA transcriptase inhibitory activity (I%) of the sample is calculated using the following formula.

For shaking culture to produce levistin, put 100 rrtl of culture solution into a 500 rrt flask, and add 27 to 2
The experiment was carried out on a shaker at 9° C. and 130 reciprocations for 4 minutes at an amplitude of 8 cIIL.

Using Streptomyces E397-1 strain, various liquid media with nutrient compositions shown in Table 1 below were prepared in advance using E.
A culture solution cultured with shaking in a medium at 27''-29°C for 3 days was used as a seed and inoculated at 1 rr Lt\, and after 4 days of culture, the activity of levistin produced in the medium was measured.

E medium, Eg, ff medium (a medium in which the carbon source glycerin of E medium is replaced with glucose), and SGF medium are optimal for production, and when revistin in these culture solutions is measured using the above assay system, it is found that Rev. The concentration that inhibits enzyme activity by 50% is 1/100 to 1/100 of the culture broth.
It was sufficient to dilute and add 1.3/100.

Even when sodium thiosulfate, manganese chloride, and cobalt chloride were added to the E medium, there was almost no effect on increasing production, or rather it was only slightly reduced.

The soybean flour F is Nikka Meat from Nichika Yushi KK, and the soybean flour S is 83-S-L from Ajinomoto KK.

In the above Table 10M-series medium, GF-medium 1X-medium, revistin was produced only 60 to 10 times more than when E medium was used.

Lepistin is well preserved in water, and in the culture solution of its producing strain E397-1, lepistin, which is mainly present in the liquid part, is substantially not extracted by butanol, ethyl acetate, ether, chloroform, benzene, etc.

Treatment with these solvents can be used if necessary to remove impurities.

Levistin in a culture filtrate or other aqueous solution, from which solids such as bacterial cells have been removed by filtering or centrifuging the culture solution, can be precipitated by salting out or by adding a suitable precipitant.

A preferred example of salting out is a method using ammonium sulfate saturation.

Furthermore, since levistin is an acidic substance, it can be precipitated by adjusting the pH of an aqueous solution containing levistin to 3 or less.

Cetyl and pyridinicum chloride, which are known as precipitants for acidic polysaccharides, are preferred examples of precipitants.Also, to collect revistin, anion exchange resin, anion exchange cellulose, etc. are used depending on its acidic properties. is available.

In this case, Dowex-1 (strongly basic anion exchange resin such as Dow Chemical Co., Ltd.), Anopalite IR-4B (
Weakly basic resins such as Rohm and Haas Co.
It can also be collected using AE+, ECTEOLA-, and cellulose (manufactured by Plakun, etc.).

It is also possible to collect levitin from an aqueous solution of levitin by adsorbing it on activated carbon and eluting it with 50% thiacetone water.

Levistin (product of Example 3 below) is an acidic substance in the form of a white or light brown powder, with no clear melting point.
Decomposes gradually at temperatures above 0°C.

Virtually intoxicating with butanol, ethyl acetate, ethyl ether, chloroform, and benzene when exposed to water.

The specific optical rotation is [α″jD7−60° (0,005 degrees, water)
It is.

Adsorbed by strongly basic anion exchange resin and activated carbon.

Levistin is a high molecular substance estimated to have a molecular weight of 2.5 to 3 minutes.

Terminal absorption was observed in the ultraviolet absorption spectrum.

The infrared absorption spectrum (KBr) of Levistin is shown in the accompanying drawings.

Levistin is heat stable and has a pH of 100 in the range of 3 to 10.
Stable when heated at ℃ for 20 minutes.

Judging from the results of electrophoresis, gel filtration, ultracentrifugation, and limit filtration, levistin is an acidic polymeric substance.

The color retention of Levistin is as follows.

Positive: Anthrone-sulfuric acid (green), platysma, myron (reddish-brown precipitate), ninhydrin (purple-pink) reaction.

Negative: Mauritsch, Fehring, Pauli, Büllets, xanthoprotein reaction.

Regarding the physiological activity of levistin, the above-mentioned crude lepistin powder inhibits the activity of Wuers transcriptase by a factor of 50 at 23 μEl/rrLL.

This 50-degree inhibitory concentration is similar to that of other antibiotics such as N-demethylrifampicin (N-demethyl-rifampicin).
200 tt &/ml of f-ampicin, 24μ of bleomycin,
/J, indicating that Levistin has a strong inhibitory effect on Vase transcriptase.

Staphylococcus aureus ATCC6538p, Bacillus subtilis ATCC6
633 Candida albicans
bicans 3147) s) Trichophyton mentagrophytes (Trichophyton m.
e-ntagroplytes 640 Pseudomonas aeruginosa,
Escherichia coli, NIHJ Plasmodium ATCC607, Bacillus
Xamthomonas ci
tri) shows no antibacterial activity at 10 μg/ml.

Even when injected intravenously into mice in physiological saline, 500
rILVk19 body weight amount does not cause acute toxicity.

Levistin is a murine leukemia virus called Rausc.
her leukemia virue (RLV
) also shows the effect of suppressing the tile formation caused by RLV infection.

The spleen tumors seen early after RLV infection in mice are associated with lesions of erythroblast proliferation rather than leukemia, but are usually used as indicators in RLV infection and treatment experiments.

The present inventors also used this splenic tumor as an indicator to determine the RL of lepistin.
The therapeutic effect on V infection was investigated.

Using the crude Levistin powder obtained in Example 1, Rau
scher leukemia virus (RLV
) The therapeutic effect on infected mice was investigated.

In experiment 1, the post-infection virus was prepared by grinding up the tiles of a mouse 4 weeks after RLv infection, and this was mixed with physiological saline for 6 hours.
0.5m of virus solution diluted 40 times! Infection was established by injecting into the peritoneal cavity of mice.

As shown in Table 2a, the infection treatment group (v1R+) is the first group in which Bistin was injected intraperitoneally for 4 days from the day of infection, and the group ■ in which bistin was injected 5 days after infection. A control group (V-R1) in which only the drug was given without virus infection was placed in each group.

There was also a control (VR) that was infected but not administered any drug.

After 12 days, all mice were sacrificed and the lungs were separated and weighed.

There were 4 animals in each group, and the lung volume was determined. It was defined as inhibition rate (aA).

The denominator is the enlarged part of the lung due to the effect of the virus only;
The molecule is the effect of the mutual interaction between the virus and the drug, excluding the effect of the drug from the increased portion of the tile in the table.

The test results are shown in Table 2b.

・As is clear from the results in Table 2b, 8rr in the first group
Lg/mouse/day and 4rrLg/max day showed inhibition of about 40%, but 2m9/mouse/day had no effect.

In group Ⅰ, 8rrLg showed about 50% inhibition and 4rng showed strong inhibition.

2rn9 was also effective.

According to these results, it appears that treatment with Levistin is more effective if started at a later stage than in the early stages of infection.

Next, in Experiment 2, the infectious dose of RLV was reduced to 1000
The double diluted virus solution was made into 0.5 mt.

As shown in Table 3a below, the animals were divided into Group 1, in which the drug was administered for 11 days from the day after infection, Group 2, in which the drug was administered for 6 days from the next day, and Group 2, in which the drug was administered for 6 days starting 6 days after infection.

In this experiment, a blocking effect was seen only in group Ⅰ, which started treatment late.

The test results are shown in Table 3b.

From the above experiments, it was found that administration of Vvistin inhibited the increase in lung weight due to viral infection.

%, a clear inhibitory effect was observed when Levistin was administered 6 days after virus infection.

'This fact indicates that Levistin can be used as a cancer treatment drug or an adjuvant for cancer treatment against virus-induced carcinogenesis.

Currently, rifamycin derivatives (S
-S, Yang et al., J - Nat'tCancer
In5t, 4 'j, '7 and '61 (1
972)) and polynucleic acids ('R-J-Er1cks
on et al., B.B.

R, C, 52, 1475 (1973), S, K.

Arya et al., B, B, R, C59,608 (19
74)') is known.

As is clear from the strong inhibitory effect of revistin as described above, revistin is a high-molecular substance, and rifampicin derivatives are low-molecular substances, so they are different.

Furthermore, since levistin migrates to the heptanoyl layer upon phenol extraction, it is clear that it is not a polynucleic acid or a nucleic acid.

Therefore, Levistin was confirmed to be a new inhibitor of Rivas transcriptase.

In addition, the Levistin powder obtained in Example 3 below showed a single band when stained with Commergy Brilliant Blue in 15% polyacrylamide gel electrophoresis (Tris-glycine buffer, pH 8,6). Therefore, we performed the following elemental analysis, amino acid analysis, and sugar analysis.

The results of elemental analysis are C47.04, N6.50 and N9.
．． 96%, sulfur and...rogen free, ash content 1.7%
Met.

For amino acid analysis, the above Levistin sample was acid-thawed in 6N hydrochloric acid at 110°C for 16 hours, and amino acid analysis was performed. Lys 1.75 * Hisl, 20
．． Arg3.06. Asp 5.10. 'I'hr2.
97,5er4.23. Pro 3.68. Gly2.
74. Al a 3.05. Gys O,24,V
a 13.54* Me t O, 78y ILeu
1.4L Lev2.90. TyrO,89,Phe
It was 1.73% (42.28% of total amino acids).

Next, for sugar analysis, the sample was hydrolyzed with IN sulfuric acid at 100° C. for 9 hours, acetylated with pyridine acetate anhydride, and then analyzed for sugar content by gas chromatography.

As a result, the W/W% was glucose: 3.26%, galactose: 2.09%, mannose: 4.01%, and trace amounts of rhamnose and xylose were also observed.

Furthermore, when the infrared absorption spectrum (KBr,') of this sample was measured, the graph shown in the attached drawing was obtained.

Using this sample, we investigated the effect on the transformation of normal rat kidney cells by the RNA tumor virus using tissue culture methods, and found that adding revistin to the culture medium immediately after infecting the cells with the virus resulted in a very significant effect as shown below. In addition, it blocked cell transformation (tumor formation) caused by the virus.

That is, 100% at 50°C mg/rrt, 25°C m
The inhibition was 83% at g/rat and 65% at 10 μg/rILt.

Furthermore, it was found that addition of the medium rlevistin 24 hours after virus infection inhibited transformation to a considerable extent as shown below.

That is, 75μg/rrLt is 4556. ．． 50μg/
It was inhibited by 35% with at and 5% with 25 μg/rrLt.

The above results indicate that revistin can be expected to have a preventive effect when considering the involvement of viruses in human cancer.

Hereinafter, examples regarding the production and collection of levistin will be shown.Since the properties of levistin, its production method, and its extraction and purification method have been clarified by the present inventors, its collection will be carried out based on the knowledge shown in this specification. The method also includes modified methods and is not limited to the examples.

Example 2.5% L/Butomyces E397-1 strain (Feikoken Bacterial Serial No. 2869) that produces rubistin
, glycerin, Part 1 polypeptone, 0.2% yeast extract, 0.6%, and inoculated into a medium containing 27%
Shaking culture was carried out at ~29°C for 4 days.

Thereafter, the culture solution was heated at 10°C for 10. The mixture was subjected to centrifugal sedimentation at 000 rpm to remove solids including bacterial cells.

Add ammonium sulfate (2161) to saturation to the obtained supernatant culture solution (3 t), stir at room temperature for 20 minutes, and then gently stir at 4° C. overnight.

The precipitate was collected by centrifugation at 4° C., 1000 Or, pm, 10 minutes, and dissolved in distilled water (100 m, ff).

The insoluble portion is extracted again with distilled water (100rrLt).

The liquid layers were combined and the dialyzed solution was freeze-dried for 24 hours against distilled water (5,/, x5 times) to obtain a crude powder of 3 (Bi').

Inhibition of 50% of Livane transcriptase activity (
ID.

) was 4.6 μg (yield 87%).

This powder was dissolved in 125 m, ff of distilled water, kept at 20°C or higher, and an excess of 10% cetyl pyridinium chloride solution (150 rrLa) was added, and the resulting precipitate was heated at 20°C.
The mixture was collected by centrifugal sedimentation at 10.00 rpm for 10 minutes, washed sequentially with distilled water (30771tx2) 24M, 4.8M saline (3 om each, +g), and then poured into a 0.IN caustic soda solution (lOOmt) in VCC.

This m solution was kept at 20°C or higher to 0. Dialysis was performed for 4 to 5 hours against IN caustic soda solution (14 x 2), distilled water (1 t x 2), and then distilled water (5 t x 3).
Dialysis was performed overnight at 4°C.

This dialysis fluid was freeze-dried to produce Revistin crude powder (brown e).
(1-5Fs ID50: 2.3μ, 9/a yield 8
Section 7) was obtained.

Example 2 2.5 parts in a jar fermenter with a capacity of 30 liters,
Glucose, 1%, polypeptone, 0.2% yeast extract,
Medium containing 0.6% calcium carbonate (' pH 6,
8) was added, sterilized, and cooled to 27°C.

Then, in contrast, Streptomyces E397-1
The culture song OOm, l obtained by culturing the strain (Feikoken Bacillus No. 2869 with shaking for 2 days in the same medium) was planted, and the aeration volume was 15 tons.
/min, stirring at 200 revolutions/min, and culture at 27°C.

After 72 hours of culture, the culture solution was centrifuged to remove solids including bacterial cells to obtain a culture filtrate with a pH of 7.2.

This was precipitated with ammonium sulfate (9.5 kg) and dialyzed in the same manner as in Example 1, and the dialyzed solution was freeze-dried to obtain 14.9 g of crude revistin powder.

The D5o of this powder was 8.2 μgml, and the yield was 85%.

Example 3 Streptomyces E3 obtained under the same conditions as Example 1
Bacterial cells were removed from the culture solution of the 97-1 strain (Feikoken Bibori No. 2869) to obtain 8400rrLt of supernatant culture solution.

Add 2 ky of ammonium sulfur to this and stir gently overnight.

The precipitate was collected by centrifugation at 1000 rpm for 10 minutes at 4°C and dissolved in distilled water.

At this time, in order to reduce the amount of liquid as much as possible, a small amount of distilled water was added, the pH was adjusted to 7 to 8 with IN sodium hydroxide solution, and the mixture was centrifuged to obtain a supernatant.

For precipitation, repeat the same operation and finally add 118 ml of distilled water.
By adding rrLt and 23 rrLl of sodium hydroxide, almost all the precipitate was dissolved (total amount 220 rrL7).

This solution was added to a Sephadex G-750 column (5 x 15
970ml of the above extract (220m4) per K1 time
was injected and developed with 0.025N sodium chloride M solution to perform column chromatography.

The mixture was separated by 10 m, and the 160th and 200th portions were dialyzed against distilled water for 50 hours and freeze-dried to obtain 6.9 g of crude powder (I).

This powder α) was added to 0. OIM) Lys buffer (pHs, 1)
A Sephadec kG-75 column (5 x 125m) was dissolved with 70rrttK and treated with the same buffer and developed with the same buffer.

A portion of 10 m4 was collected and the 200th and 240th portions were dialyzed again against distilled water for 45 hours and freeze-dried to yield 1.68. ! A crude powder of No. 9 was obtained.

This is 0. OIM) Lys buffer (pH 8,1) 10
Ultrogel (
LKB Co.) AcA54 column (2X80crn) was developed with the same buffer solution, and aliquoted in 5 m/: 40
A portion eluted from 50 bottles 1 was taken, dialyzed against distilled water for 30 hours, and lyophilized to obtain revistin as a powder of 0.92.

The ID of this powder is 61.3 μg/rnl, and the yield is 4
I was in Section 9.

This Levistin powder gave a single band when electrophoresed on a 15% polyacrylamide gel.

[Brief explanation of drawings]

The attached drawing is an infrared absorption spectrum (KBr tablet) of Levistin powder obtained in Example 3 of the present invention.

Claims (1)

[Claims] 1. A method for producing bisutin, a liguarth transcriptase inhibitor, which comprises culturing a lepistin-producing bacterium belonging to the genus Streptomyces, and collecting levistin, a liguarth transcriptase inhibitor, from the culture solution.