JPH01228495A - Enzyme inhibitor, its production and use - Google Patents

Abstract

PURPOSE:To produce peptide which is used to inhibit the development of gastric ulcer by culturing Actinomycetes SAM-0966 in a medium. CONSTITUTION:As a result of soil search and isolation, is obtained Actinomycetin SAM-0966 strain (A) having following properties: morphology, forming aerial mycelia of 0.8-1.0micrometer diameter; culture finding, the aerial mycelia is pale white in an agar medium containing sucrose and nitrate, back face is white to pale yellow, no soluble pigment; growth optimal temperature is 22-30 deg.C; assimilating D-glucose, but not inositol; and producing a substance to inhibit the activity of carboxylproteinase, cysteine proteinase and serine proteinase. The strain A is cultured in a medium containing glucose or the like at 4-7pH, at 25-35 deg.C for 1 to 2 days to obtain a culture mixture. Then, component B is extracted and purified to obtain the peptide, thyrostatin, an enzyme inhibitor, of the formula, having a molecular formula: C29H39O6N3, 525.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel peptide compound that exhibits enzyme function inhibitory activity of carboxyl proteinase, cysteine proteinase, and serine proteinase, and its production method and uses.

(Prior art and problems) Pepstatin (Umezawa et al., J-Antibiotics,
23, p. 259, (1970), or JP-A-47-29582), or 5P-1 (Murao et al., A
gric, Biol.

Chem, vol. 34, p. 1265, (1970)),
or pepstatin (Miyano et al., J. Antibioti
cs+ vol. 25, p. 489 (1972), or JP-A-88281-1983). These pepstatin analogs include hydroxypepstatin in which the alanine residue is replaced with a serine residue (Umezawa et al., J.
, Antibi.

tics, vol. 27, p. 615, (1973)),
Linear or branched fatty acids with N-terminal acyl groups ranging from acetic acid with a 2-carbon chain to alginic acid with a 20-carbon chain (Aoyagi et al.
J, Antibiotics+ vol. 26, p. 539,
(1973)) have also been separated. Pevcinostreptin (Kakinuma et al. J, Takeda R
es, Lab, vol. 35, p. 123, (197
6)) is also the same substance contained in these pepstatins. All of these compounds are characterized by being pentapeptides containing the unusual amino acid statin ((3S,4S)-4-amino-3-hydroxy-6-methylhebutanoic acid) at the third residue from the N-terminus and at the C-terminus. (In the case of pepstatin, stanone ((3S)-3-amino-5-methyl-hexan-2-one) is attached to the C-terminus) and a linear or branched fatty acid is acyl bonded to the N-terminus. Many of the conventionally known low molecular weight peptide inhibitors of carboxyl proteinase are produced by microorganisms, and they are basically all analogs of pepstatin. In addition, some lupoxyl proteinases are inhibited by pepstatin analogs (e.g., Murao et al., Agric, Biol, Chem,
t vol. 36, p. 1647, (1972), Murao et al.
and their 1nhibitors” (
Kost Kalv, ed.), 379-3.
Page 99, Walter de Gluyter
& Go,t Berlin+ New
York, Oguni et al., Agric, Biol,
Chem, + vol. 51, p. 3073, (198
7), or Oguni et al., Biochim, Biop
h7s, Actat volume 923, page 463, (1
987), etc.), and of course does not show any inhibitory activity against cysteine or serine-based neutral proteinases.

Furthermore, among the conventionally known low molecular weight peptide inhibitors against cysteine proteinase, a typical example is leupeptin (Shimizu et al., J., Antibiot,
, Vol. 25, p. 515, (1972)), Strepin (
Ogura et al. N Agric, Blol, Chem,...
Volume 49, page 799, (1985), or Japanese Patent Application Publication No. 1983
-28990, or stuccobin (*Fuji et al., Agr.
ic, Biol.

Chem, + vol. 51, p. 861, (1987
), or JP-A-81-106600). These compounds strongly inhibit the activity of cysteine or serine brotinase, but do not show any inhibitory activity against carboxyl brotinase.

From this point of view, carboxyl proteinases are not pepstatin analogs, but have a new structure,
There is also a need for physiologically active substances that exhibit strong inhibitory activity against pepstatin-insensitive enzymes. Furthermore, if such a compound also exhibits an inhibitory effect on cysteine or serine proteinase, its range of applications would be infinitely expanded, but low molecular weight peptide inhibitors that exhibit such a broad inhibitory spectrum are currently lacking. Not known to anyone.

(Means for Solving the Problems)) Therefore, the present inventors have discovered that the present invention has a novel structure from nature and exhibits enzyme function inhibitory activity not only for carboxyproteinase but also for other cysteine and serine proteinases. We conducted an intensive search to find new microorganisms that produce physiologically active substances. As a result, the culture solution of actinomycetes isolated from the soil of Hachigamine, Sakai City, surprisingly produced many physiologically active substances that exhibited enzyme function inhibitory activity against carboxybrotinase and cysteine proteinase. I found it. These compounds of the present invention differ from conventionally known pepstatins in terms of their constituent amino acids, sequences, and C.
They differ greatly in that they have an aldehyde group at the end.

(Structure of the Invention) The proteinase inhibitor of the present invention is represented by the following formula (I). In addition, a novel microorganism SAM-0966 [Feikoken Bacterial Serial No. 9927] that efficiently produces the peptide compound of the present invention
FERMp-9927) has the following mycological characteristics.

1. Morphological findings The SAM-0966 strain has a diameter of 0. 8-1. 0)tm
Forms aerial mycelium. Aerial hyphae are branched, and branched hyphae may be significantly short. Coremia with a diameter of 2 to 4 PTn is formed in the aerial hyphae.

A few spherical bodies were observed.

SAM-0966 strain was tested on plain agar, 1/10 yeast starch agar, 1/20 V-8 youth agar, oatmil agar with 0.1% yeast extract, and on the medium used to observe culture findings. When cultured at 28°C for 117 days, no formation of spores, sporangia, sporangia, or sclerotia was observed.

2.Culture findings (cultured at 28°C for 17 days) Muslocucle/nitrate agar medium: Aerial hyphae, faint, white, color on back side, white to pale yellow, soluble pigment absent.

Muglucose-asparagine agar medium: Aerial mycelia, none, color of back side, dark brown, soluble pigment, yellow.

Muglycerin/asparagine agar medium: Aerial mycelium, none, color of back side, reddish brown, soluble pigment, brown.

Mustarch/inorganic salt agar medium: Aerial mycelium, thin, white, color on the back, grayish brown, soluble pigment, none.

Mutirosin/agar medium: aerial hyphae, medium, white to gray,
Underside color, dark brown, soluble pigment, brown.

Nutrient agar medium: Aerial mycelia, none, back side color, pale yellow, soluble pigment, pale yellow.

Yeast/malt agar medium: Aerial mycelium, blue, white ~
Gray, underside color, tan, soluble pigment, none.

Mutual oatmeal/agar medium: Aerial mycelium, white to gray, underside yellow, soluble pigment absent.

3. Physiological findings ■Growth temperature range: 17°C using yeast extract/glucose liquid medium
19°C122°C124°C127°C130'C1
33°C135°C138°C, 42°C145.5°C
As a result of tests at temperatures of , and 49°C, growth was observed at temperatures below 35°C. The optimum temperature for growth seemed to be around 22°C to 30″C.

■ Liquefaction of gelatin (28°C) Glucose-peptone-gelatin medium Negative simple gelatin medium Negative broth gelatin medium
Negative ■ Hydrolysis of starch
Positive■ Coagulation of skim milk (28°C)
Negative■ Peptonization of skim milk (28℃) Negative■
Production of melanin-like pigment Peptone/yeast/iron agar medium Negative tyrosine/
Agar medium Negative Tryptone Yeast Agar medium Negative ■ Nitrate reduction
Positive ■ Availability of carbon sources (Bridham-Gotlieb agar medium, cultured at 28°C for 117 days) D-glucose ++ D-xylose + L-arabinose ± L-rhamnose - D-fructose Raffinose - D-mannitol Inositol - Sucrose − However, ++: Strongly use it, +: Use it, ±: I doubt whether it will use it, 1: Do not use it.

4. Chemical properties ■ 2.6-diaminopimelic acid Whole bacterial cells and cell wall hydrolyzate were added to 5taneck+
J.L. and Roberts*G.D.
Method (ApI) of Lied Microbiol
As a result of an investigation in accordance with OGY+ Vol. 28, p. 22θ, 1974), the existence of two isomers, meso-2゜6-diaminopimelic acid and L,L-2,6-diaminopimelic acid, was observed. .

■Sugar The presence of ribose, mannose, glucose, and galactose was observed in the hydrolyzate of whole bacterial cells. Furthermore, the presence of mannose and galactose was observed in the cell wall hydrolyzate.

(2) Contains quinone MK-9 (-H8) and MK-9 (H6t) as main components.

It is believed that the compound (I) of the present invention can be easily produced by those skilled in the art by a known peptide synthesis method, and compounds obtained by this synthesis method are also included in the present invention. A practical production method is to culture actinomycete SAM-0966, which has the ability to produce the physiologically active substance of formula (I), in an appropriate medium, and to separate and purify the culture.

The medium used in producing the substance of the present invention may be liquid or solid, but shaking culture or aerated agitation culture using a liquid medium is usually convenient. Any medium may be used as long as the microorganisms producing the substance of the present invention grow and the substance of the present invention is accumulated in the medium. That is, carbon sources include, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic acids, etc., and nitrogen sources include, for example, peptone, protein hydrolysates such as casamino acids, meat extracts, and yeast. Extract, soybean meal, cornstarch liquor, amino acids, ammonium salt,
Nitrates and various other organic or inorganic nitrogen compounds are used. As inorganic salts, phosphates, magnesium sulfate, and sodium chloride may be added, and vitamins, nucleic acid-related compounds, etc. may be added for the purpose of promoting bacterial growth. Note that adding antifoaming agents such as silicone, polypropylene glycol derivatives, and soybean oil to the medium may be effective in increasing the amount of the present substance accumulated.

In culturing, conditions such as culture temperature, culture period, culture liquid, etc. are appropriately selected and adjusted so as to maximize the accumulation of the substance of the present invention.
Cultivation may be carried out at 5°C to 35°C for 11 to 2 days, and the pH of the medium is preferably maintained at pH 4.0 to 7.0.

By culturing in this manner, the substance of the present invention is produced and accumulated in the culture. When culturing using a liquid medium, the target substance is mainly accumulated in the liquid part, so it is separated from the filtrate or supernatant after the culture is filtered or centrifuged to remove bacterial cells. However, it is also possible to separate the target product directly from the culture solution, if necessary, without removing the bacterial cells. Various means based on the chemical properties of the substance of the present invention may be employed to separate and purify the target product from the culture. That is, for example, precipitation by adding a precipitant such as ammonium sulfate, extraction with an organic solvent such as n-butanol that is not arbitrarily mixed with water and which dissolves the substance of the present invention, and highly polar solvent such as methanol or ethanol. removal of impurities by treatment with hexane, gel filtration using Sephadex, ion exchange chromatography using various ion exchangers such as ion exchange resin, ion exchange cellulose, and ion exchange Sephadex, activated carbon, alumina, silica gel, Adsorption chromatography using an adsorbent such as Amberlite-XAD-1゜2 or crystallization from a solvent can be effectively used, and by using an appropriate combination of these methods, the substance of the present invention can be produced as a colorless needle. It is isolated in crystalline form. However, methods other than these can be used as appropriate as long as they effectively utilize the properties of the substance of the present invention. A particularly preferred adsorbent is Diaion HP
-20, Sephadex LH-20, TSKG-300
081 Cosmocil 10C18, and DEAE-cellulose combinations.

As shown in the Examples, the physiologically active substances produced according to the present invention exhibit inhibitory activity not only against aspartic proteinases such as pepsin, but also against pepstatin-insensitive carboxyl proteinases. It also showed strong inhibitory activity against cysteine proteinases such as ficin and ficin.

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.

(Example 1) A. Production of peptide compound tyrostatin by culturing actinomycete SAM-0966.

A pure culture of actinomycete SAM-0966 was inoculated into a synthetic medium (pH 6.0) consisting of 0.5% polypeptone, 1.0% glucose, 0.5% meat extract, and 0.3% sodium chloride. and incubate at 30°C in a 30-capacity incubator.
Aeration and stirring culture was carried out for 24 hours at an aeration rate of 20 L/min and 300 revolutions/min (Fig. 1). Centrifuge the culture and remove the supernatant18.
2 (pH 4,8) under reduced pressure and water bath temperature 40~
50°C) to about 1/10 the capacity. Equivalent amounts of ethyl acetate were added to this concentrated solution to perform extraction three times, and the ethyl acetate layer was concentrated under reduced pressure and dried. The recovery rate from the culture supernatant was 48%.

This crudely purified inhibitory substance was dissolved in 200 ml of methanol,
Sephadex LH-2 equilibrated with methanol in advance
0 column (30 mm x 550 mm). The column was developed with methanol, the eluate was fractionated into 7.8 ml portions, and the inhibitory activity of each fraction was measured. The inhibitor was eluted as a single peak. (Figure 2) The recovery rate from the culture supernatant was 30%.

Since the inhibitor solution obtained above still contained colored substances, activated carbon treatment was performed to remove them. Decolorization treatment was performed first with activated carbon at a weight/volume ratio of 1% and then with activated carbon at a weight/volume ratio of 0.5%. This operation removed most of the colored material. The recovery rate from the culture supernatant was 21%.

430 ml of the inhibitor solution obtained above was concentrated under reduced pressure to obtain a slightly yellowish white powder. This was dissolved in ethyl acetate and adsorbed on twice the weight of silica gel, which was then equilibrated with hexane in advance on a silica gel column (20 mm).
X 30011111 = 20 times the weight of silica gel)
Loaded on top. The column was first washed with hexane and the adsorbed inhibitors were eluted by stepwise increasing the concentration of ethyl acetate in hexane. (Figure 3) As a result of collecting the active fractions and measuring the inhibitory activity, the recovery rate from the culture supernatant was 10%.

The inhibitor solution thus obtained was concentrated under reduced pressure, then dissolved in a small amount of methanol, and the solution was allowed to stand at room temperature to obtain about 100 mg of colorless needle crystals. The recovery rate from the culture supernatant was 5%.

As described above, tyrostatin (Tyr
ostatln).

B8 Physicochemical properties of the peptide compound tyrostatin of the present invention.

Molecular formula: C25HssOaN3 Molecular weight: 525.3 Properties: Colorless needle crystals Melting point: 125-135°C (decomposed) Solubility: Easy to dissolve Soluble in methanol, N, N-dimethylformamide, dimethyl sulfoxide Ethanol, n-butanol, acetone, Ethyl acetate, poorly soluble in acetonitrile, diethyl ether, petroleum ether, chloroform, benzene, carbon tetrachloride, n-hexane Specific optical rotation: [:αl','=-43. 3° (c
=0. 13) Mass spectrometry: (FABMS) 52
E3 (M4+1) Infrared absorption: 3350, 3320
．． 1?40°1620, 1542. 1523° 1250 (am ultraviolet absorption: 280 nm (no notable absorption in 250-400 nm other than 280 nm ultraviolet absorption) 1) (Nuclear magnetic resonance absorption (TMS standard, in δ-dimethyl sulfoxide solvent): Chemical shift ppm 4, 16 (LH, m) (For each chemical shift in the table above, the number of IH from which the signal originates, bond/, s-turn, and binding constant are written in 'Ax units.) I30 Nuclear magnetic resonance absorption (TMS standard): 21.8, 2
2.1, 22.1, 22.9.24.0, 25.
5, 32.5, 36.4.41.1, 44.5,
50.9, 54.0.59.9, 115. 0
(2C), 115.2 (2C), 127.6.12
8.3, 130.3 (2C), 130.4 (2C), 156.0.15B, ・2
, 171.9, 171.9.172.8, 200
．． 9 (1) pm) Color reaction: Positive; 2,3.5-) Riphenyltetrazolium chloride reaction, 2,4-dinitrophenylhydrazine reaction,
Chlorine-tolidine reaction, Thorin reaction, Dragendorff reaction, Pauli reaction negative; ninhydrin reaction, Sakaguchi reaction, Erlich reaction elemental analysis; 1.305 64.71 7.58 7,771
．． 252 64.27 7.49 7.71 Theoretical value 8E3,25 7,48 8.00E1 Amino acid analysis of this compound Dissolve 500 μt of this compound in 4 ml of methanol,
Dispense 1 ml of each into test tubes and dry. Add an appropriate amount of 8N hydrochloric acid to this test tube, vacuum seal it, and heat it to 105°.
Hydrolyzed at C for 24.48 and 72 hours, respectively.

After hydrolysis, they were dried under vacuum, and 1 ml of 0.02N hydrochloric acid was added to each to dissolve them. When this solution was diluted 3 times and subjected to amino acid analysis, leucine and tyrosine were detected. ◇ After considering the molecular weight of this compound and the molar ratio of its constituent amino acids, the following values were obtained. .

The analysis results are shown below.

From the above analysis results, it was determined that the structure of the compound tyrostatin in the present invention is represented by the following formula (I).

(Example 2) Enzyme inhibitory activity of the compound of the present invention The enzyme inhibitory activity of the substance of the present invention was examined by the following measuring method.

(a) From Pseudomonas sp., N0110
Enzyme inhibitory activity against 1 carboxyl proteinase (PCP) and N00T-22 carboxyl proteinase (xCP) derived from Xanthomonas sp. was determined by the following method.

First, proteinase activity was measured using the casein-folin method (Ca
A modified method of Sein-Folin) was used.

1.33% hammersten casein solution (0.05M lactic acid buffer, pH 3.0) 1. Add 0.0 mL of enzyme solution diluted to an appropriate volume to 5 mL. After 15 minutes of reaction at 37° C., 2 ml of 0.44 M trichloroacetic acid solution was added to stop the reaction. Precipitate formation of undigested casein is 37
After 20 minutes at °C, add 0.5 ml of filtrate to 0.44 M sodium carbonate solution. 5 ml, Folin reagent 0. 5ml
was added and the color was developed at 37°C for 20 minutes, and then the absorbance was measured at 60 nm. A blind test was performed by reversing the order of addition of the enzyme and precipitation reagent. 1 enzyme unit (IPU) of proteinase activity that shows a color corresponding to tyrosine 1γ in 1 minute
And so.

The inhibitor activity is according to the above proteinase activity, and the enzyme solution is 0. 5+I11 and 0.01ml of the inhibitor solution of the present invention
After shaking at 37°C for 5 minutes, substrate 1. Add 5 ml and perform an enzyme reaction for 15 minutes, and compare with the control (add methanol instead of the inhibitor of the present invention) to determine the inhibitory ability (
The 50% inhibitory concentration (5IC6iI) was determined. However, since no linearity was observed between the inhibition rate of carboxyl proteinase (PCP) derived from Pseudomonas sp. and the concentration of the inhibitory active substance, a calibration curve was created. In actuality, the dilution was performed so that the inhibition rate was around 50%, and the inhibitory activity was determined from a standard curve. The amount of inhibitor that inhibits 30 PU of enzyme by 50% is defined as 1 inhibitor unit (IIU).
).

In addition, the inhibitory activity against pepstatin-sensitive aspartic proteinases such as pepsin derived from pigs and carboxyl proteinases other than the above two proteinases was determined by the same method as above, but with a reaction time of 10
It was a minute.

(b) Inhibitory activity against cysteine proteinase such as papain was determined by the following method.

Enzyme solution diluted to an appropriate amount (0.05M Tris-HCl buffer solution, pH 7.5) 0. 4 ml of 0.05 M cysteine and 0.02 M EDTA aqueous solution Q, 1
ml and shaken at 37°C for 10 minutes.

To this solution was added 10 μm of an aqueous solution of the inhibitory substance of the present invention, and the mixture was further shaken at 37° C. for 5 minutes. An additional 1°33%
Casein solution (0.05M) Lis-HCl buffer solution, PH7
,5) 1. Add 5 ml and react for 10 minutes at 37°C, then 2 ml of 0.44M)! The reaction was stopped by adding J chloroacetic acid solution. Precipitation of undigested casein occurs at 37°C.
The absorbance of the filtrate at 280 nm was measured. The inhibitory ability (50% inhibitory concentration: IC5) was determined by comparison with the control (add methanol instead of the inhibitor of the present invention).
a) was obtained.

(C) The inhibitory activity of bovine-derived α-chymotrypsin against serine proteinase was determined by the following method.

The inhibitor activity was determined according to the method for measuring carboxyl proteinase inhibitory activity in (a). After shaking 5 ml and 10 μm of the present inhibitor solution at 37°C for 5 minutes, substrate 1
．． 5 ml was added and an enzyme reaction was performed for 10 minutes, and the inhibitory ability (50% inhibitory concentration: IC5-) was determined by comparison with a control (methanol was added instead of the inhibitor of the present invention). However, in the case of subtilisin, a 0.05M sodium carbonate-sodium borate buffer solution (pH 9,5) is used as the buffer solution instead of the lactate buffer solution, and in the case of trypsin and chymotrypsin, a Tris-HCl buffer solution (pH 9.5) is used as the buffer solution.
H7,5) was used.

(d) Inhibitory activity against metalloproteinases such as thermolysin was determined by the following method.

The inhibitor activity was determined according to the method for measuring carboxyl proteinase inhibitory activity in (a). After shaking 5 ml of this invention inhibitor solution 10771 at 37°C for 5 minutes, substrate 1. Add 5 ml and perform enzyme reaction for 10 minutes, then control (
Methanol is added instead of the inhibitor of the invention. ), the inhibitory ability (50% inhibitory concentration: IC5s) was determined. However, the buffer solution in this case was a Tris-HCl buffer solution (pH 7.5).

50% inhibitory concentration for typical proteinases: IC
59 is shown in the table below.

IC611 (μM) In addition to these enzymes, the compound of the present invention can also be used in combination with morsin, which is a pepstatin-sensitive carboxyl proteinase, Aspergillus niger var, type B enzyme derived from Macrosphorus, enzyme derived from Rhodotorula glutinis, and Cladosporium sp. Enzyme derived from Rhizopus
In addition to papain, it also showed inhibitory activity against ficin and promelain, which are cysteine proteinases, and subtilisin, a serine proteinase, in addition to α-chymotrypsin. However, it showed no inhibitory activity against metalloproteinases such as thermolysin.

(Effects of the Invention) Since the peptide compound represented by formula (I) of the present invention has asbaltic proteinase inhibitory activity, it can prevent or cure gastric ulcers that are thought to be caused by asbaltic proteinase. shortening the period,
It can be expected to have effects such as inhibiting granuloma growth, renal hypertension, and inhibiting the formation of viral lesions. Furthermore, since the compound of the present invention strongly inhibits some of the carboxylbroteinases known to date that are not inhibited by pepstatin, it is effective against various pathological conditions thought to be caused by microorganisms that produce these. You can also expect effects. Since the compounds of the present invention exhibit cysteine proteinase inhibitory activity, they are expected to have therapeutic effects on inflammation, cataracts, epidermolysis bullosa, diarthrosis, etc., which are thought to be caused by cysteine proteinase. Furthermore, by creating an affinity column using the compound of the present invention as a ligand, it can be used for the purification of carboxyl proteinases and cysteine proteinases, and as mentioned above, the compound of the present invention is effective against both cysteine and carboxyl proteinases. Since it is a very specific compound that exhibits enzyme inhibitory activity, its applications in biochemical and enzymological fields are immeasurable, and its application as a biochemical reagent for research on enzyme reaction mechanisms is also highly anticipated.

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes over time in the amount of bacterial cells in the culture of the present compound-producing bacteria in Examples, the inhibitor activity in the supernatant, and the pH of the medium. Figure 2 shows Sephadex L in the purification of the compound of the present invention.
It is a graph showing the elution pattern of H-20 column chromatography. FIG. 3 is a graph showing the elution pattern of silica gel column chromatography in the purification of the compound of the present invention.

Claims (5)

[Claims] (1) The following formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) A peptide compound represented by (2) Cultivate actinomycete SAM-0966, produce and accumulate a peptide compound represented by the following formula (I) in the culture solution. A method for producing a peptide compound represented by the above formula (I) by purification. (3) Actinobacteria SAM-0966
(FERMp-9927)] according to claim 2. (4) A carboxyl proteinase, cysteine proteinase, and serine proteinase activity inhibitor containing a peptide compound represented by the following formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I). (5) A microorganism named Streptomyces SAM-0966.