JPH0643436B2 - New substance trehalostatin and its production method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel substance, trehalostatin.
The present invention also relates to a method for producing the novel substance trehalostatin and an actinomycete that produces the novel substance.

BACKGROUND ART Trehalase is a type of α-glucosidase, an enzyme that specifically catalyzes the hydrolysis of the glucosidic bond of trehalose, which is widely distributed in the hemolymph of molds, yeasts, and many insects.

[Disclosure of the Invention] The present inventors have isolated Amycolatopsis (Amyc
We discovered that actinomycetes belonging to the genus O. latopsis produce a novel substance in their culture medium and cells that inhibits trehalase from insects, particularly blow flies, at extremely low concentrations, and named this substance trehalostatin.

The present invention was completed based on the above findings and relates to a novel substance, trehalostatin, obtained by culturing a trehalostatin-producing bacterium belonging to the genus Amycolatopsis and collecting the resulting culture. The present invention also provides an actinomycete strain that produces the novel substance and a method for producing trehalostatin using the strain.

Fungi that can be used in the present invention include Amycolatopsis
Trehalostatica (Amycolatopsis trehalostatica)
All trehalostatin-producing fungi belonging to the genus Amycolatopsis, including the fungi of the genus Amycolatopsis, can be used in the present invention.

In particular, the strain SAM 09, which we newly isolated from soil,
67 can be suitably used.

Amycolatopsis trehalostatica SAM 0967 has the following mycological properties:

1. Morphological findings: SAM 0967 strain forms substrate and aerial hyphae with a diameter of 0.4-0.8 μm. The substrate hyphae are branched and occasionally segmented. The aerial hyphae are branched and form chains of around 10 or more spores at their tips. The spores are 0.4-0.5 μm wide and 0.9-1.3 μm long, with smooth surfaces. No structures such as sporangia, hyphal strands, or sclerotia were observed even after 21 days of culture.

2. Culture findings (cultured at 28°C for 21 days) Sucrose nitrate agar medium: Aerial mycelium: abundant, white; reverse color: orange-brown; soluble pigment: grayish purple.

Glucose-asparagine agar medium; aerial mycelium: abundant,
White; reverse color: grayish orange; soluble pigments: none.

Glycerin-asparagine agar medium; aerial mycelium: abundant,
White; reverse color: grayish brown; soluble pigment: orange-gray.

Starch and inorganic salts agar medium; aerial mycelium: abundant, white; reverse color: whitish yellow; soluble pigment: none.

Tyrosine agar medium; aerial hyphae: abundant, reverse color: dark brown; soluble pigment: grayish brown.

Nutrient agar medium; aerial mycelium: abundant, white; reverse color: whitish yellow; soluble pigment: none.

Yeast extract/malt extract agar medium; aerial mycelium: abundant,
White; reverse color: dark brown; soluble pigment: grayish brown.

Oatmeal agar medium; aerial mycelium: abundant, white; reverse color: whitish yellow; soluble pigment: none.

Plain agar medium; Aerial mycelium: abundant, white; Back color: white;
Soluble pigments: None.

1/10 potato-carrot agar medium; aerial mycelium: abundant, white; reverse color: whitish yellow; soluble pigment: none.

1/10V-8 juice agar medium; aerial mycelium: abundant, white; reverse color: whitish yellow; soluble pigment: none.

3. Physiological findings Growth temperature range: 16°C, 19°C, and 20°C in a yeast extract/glucose liquid medium.
°C, 22°C, 25.5°C, 28.5°C, 31°C, 33°C, 36.5°C, 39.5
Test results at temperatures of 42.5°C, 45.5°C, and 47°C
Growth was observed above 19°C and below 39.5°C. The optimum temperature for growth appears to be around 28.5°C to 36.5°C.

Gelatin liquefaction (28°C) Glucose-peptone-gelatin medium Positive Simple gelatin medium Positive Broth gelatin medium Positive Starch hydrolysis Negative Coagulation of skim milk (28°C) Negative Peptonization of skim milk Positive Formation of melanin-like pigments Peptone-yeast extract-iron agar Negative Tyrosine agar Negative Tryptone-yeast extract agar Negative Nitrate reduction Positive Carbon source utilization (Pridham-Gottlieb agar,
(Cultivated at 28°C for 14 days) D-glucose + D-xylose + L-lactose + L-rhamnose + L-arabinose + D-fructose + latinose ± D-mannitol + inositol + sucrose + lactose + However, +: utilized, ± doubtful whether utilized,
-: Unutilized acid production capacity Raffinose + Inositol + D-mannitol + Lactose + Sorbitol - Erythritol + L-arabinose + Adonitol - D-galactose + [Gordon, R.E. et al. (International Journal of Syst
24, 54, 1974.] 4. Chemical properties 2,6-diaminopimelic acid was purified by the method of Staneck, JL and others. Whole cells and cell wall hydrolysates were purified by the method of Staneck, JL and others.
Roberts, GD's method (Applied Microbiology, Vol. 28, 226
As a result of the investigation in accordance with the method described in (Page, 1974), the meso-2,6-
The presence of diaminopimelic acid was observed.

Arabinose was detected in the hydrolysate of the whole cell, and galactose and arabinol were detected in the hydrolysate of the cell wall.

Quinone-based The main component is MK-9 (H4).

Contains phospholipid type phosphatidylethanolamine, but does not contain phosphatidylcholine or unknown glucosamine-containing phospholipids. This is based on the findings of Lechevalier, MP, and HA Lechevalier.
(The Chemotaxonomy of Actinomycetes, pages 227-291, In A.Dietz and DWThayer (eds.), Actinomycetes
Taxonomy,Special Publication no.6.Society for Indu
It belongs to the P-II type according to the American Society of Clinical Microbiology, USA, 1980.

Mycolic Acids The bacteria do not contain mycolic acids.

The cell wall of strain SAM 0967 contains meso-2,6-diaminopimelic acid, galactose, and arabinose.
The SAM 0967 strain forms aerial hyphae, and smooth spores adhere in chains to the tips of the hyphae. The quinone system contains MK-9 (H4) as the main component, and the phospholipid type is P-II, and does not contain mycolic acid. From the above taxonomic characteristics, the SAM 0967 strain is clearly an Amycolatopsis
It is an actinomycete belonging to the genus Amycolatopsis.
Contains one known species of subspecies. Lechevalier,MP e
t al. (International Journal of Systematic Bacteri
logy, vol. 36, p. 29, 1986), and Henssen, A. et al.
l. (International Journal of Systematic Bacteriolo
The taxonomic position of strain SAM 0967 was searched according to the method of the Japanese Society of Microbiology, Vol. 37, p. 292, 1987, and it was found to be Amycolatopsis orientalis.
alis and Amycolatopsis mediterranei are SAM 09
67 strain. Therefore, the two type strains and SA
The M0967 strain was subjected to comparative tests on its cultural properties, physiological properties, and quinone activity. The results are shown in the following table.

As shown in the table, SAM 0967 strain and Amycolatopsis orie
ntalis JCM-4600 can measure the color of aerial mycelia on yeast extract/malt extract agar medium and inorganic salt/starch agar medium.
They are clearly distinguishable from each other in terms of growth in the presence of 5% NaCl, sucrose utilization, acid production from raffinose, erythritol, and adonitol, and menaquinone composition.

In addition, SAM 0967 strain and Amycolatopsis mediterranei J
CM-4789 is clearly distinguishable from the others in terms of its aerial mycelium formation ability and menaquinone composition.

The present inventors determined that these differences were sufficient to distinguish the SAM 0967 strain from these two species. Therefore, the SAM 0967 strain was designated as a new species in the genus Amycolatposis and classified as A.
It was named mycolatopsis trehalostatica.

Amycolatopsis trehalostatica SAM 0967 was discovered in February 1989.
It was deposited domestically at the National Institute of Biotechnology, Agency of Industrial Science and Technology on February 17, 1990, under the accession number FERM P-10544, and then transferred to an international deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on February 28, 1990, under the accession number FERM BP-2784.
4) is attached.

In the present invention, the actinomycetes described above are cultured in a medium that may be either liquid or solid, but shaking culture or aeration and agitation culture in a liquid medium is usually convenient.

The medium may be any medium that allows the actinomycete of the present invention to grow and accumulate the novel substance of the present invention. That is, the carbon source may be glucose, lactose, starch, sucrose, dextrin, molasses, organic acids, etc., and the nitrogen source may be peptone,
In addition to protein hydrolysates such as casamino acids, meat extract, yeast extract, soybean meal, corn steep liquor, amino acids, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds can be used. Various inorganic salts such as phosphates, magnesium sulfate, and sodium chloride can also be added, and vitamins and nucleic acid-related compounds can also be added to promote bacterial growth. Adding antifoaming agents such as silicone, polypropylene glycol derivatives, and soybean oil to the culture medium can also be effective in increasing the accumulation of the novel substance of the present invention.

In culturing, it is preferable to first carry out a small-scale pre-culture and then inoculate the culture into the medium rather than immediately starting the main culture. For both the main culture and the pre-culture, the conditions such as the culture temperature, culture period, and liquid properties of the culture are appropriately selected and adjusted so as to maximize the accumulation of the novel substance of the present invention.
In most cases, cultivation is carried out under aerobic conditions at 25°C to 30°C, and the pH of the medium should be maintained at 5.5 to 8.0.
By culturing in this manner, the novel substance of the present invention is produced and accumulated in the culture. When culturing in a liquid medium, the target substance accumulates mainly in the liquid portion, so it is desirable to first filter or centrifuge the culture to remove the bacterial cells and then separate the target substance from the resulting liquid or pickle. However, if necessary, the target substance can be separated directly from the culture liquid without removing the bacterial cells.

When collecting the novel substance trehalostatin of the present invention as described above, the detection and quantification of the target substance can be performed by quantifying the degree to which the target substance inhibits the activity of trehalase prepared from Aleurites or pig kidney. Various methods based on the chemical properties of the novel substance of the present invention can be used to separate and purify the target substance from the culture. Effective methods include organic solvent treatment, gel filtration using Sephadex or Biogel, ion exchange chromatography using various ion exchange resins, adsorption chromatography using adsorbents such as activated carbon, silica gel, Amberlite-XAD-1 and -2, and normal phase chromatography using carriers such as YMC-PA43 and TSK-Amide 80. By appropriately combining these methods, the novel substance trehalostatin of the present invention can be isolated in the form of white amorphous crystals. However, other methods can also be used as long as they effectively utilize the properties of the novel substance of the present invention. A particularly preferred combination of adsorbents is Dowex 50WX4(H), YMC-PA43, and TSK-Amide.
There are 80 possible combinations.

The novel substance of the present invention can be used after isolation and purification, but in some cases the culture of the trehalostatin-producing bacteria can be used as is or simply after purification.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: H,H-COS of trehalostatin in heavy water
Y spectrum Figure 2: H of acetylated trehalostatin in _CDCl3
MBC spectrum Figure 3: Phase-detected NOE of trahalostatin in heavy water
SY spectrum Figure 4: N of acetylated trehalostatin in _CDCl3
OESY spectrum. Figure 5: Structures of two positional isomers of acetylated trehalostatin and their ^1H- and ^13C- spectra in _CDCl3 .
- NMR spectrum data (non-glucose portion, italicized numbers represent ¹³ C-NMR chemical shifts) [Best Mode for Carrying Out the Invention] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

Example 1: Production of trehalostatin (1) Activity measurement method The inhibitory activity at each stage of trehalostatin purification was measured as follows:
The measurement was carried out as follows.

10 μl of the inhibitor solution was mixed with 10 μl of trehalase solution (Aedes aeruginosa), and the mixture was incubated at 37°C for 5 minutes, after which it was diluted with 5 mM trehalose, 50 mM phosphate buffer (pH 6.
5) 80 μl of the mixture was added and incubated at 37°C for 1 hour. The reaction was then stopped by adding 10 μl of 50% trichloroacetic acid solution. The glucose produced in the reaction solution was quantified using the new Blood Sugar Test from Boehringer Mannheim Yamanouchi. The amount of enzyme used was such that the absorbance at the wavelength used for glucose quantification (660 nm) was 1.0 when measured according to the above method without adding an inhibitor. The enzyme (trehalase) was measured using the above method.
The concentration of trehalostatin that inhibits activity by 50% is 1 unit/
ml.

(2) Production of trehalostatin by culturing Amycolatopsis trehalostatica SAM 0967. Potato hot water extract, glucose, dry yeast extract,
Medium 3.1 consisting of monopotassium phosphate and magnesium sulfate
30 ml of pure culture of SAM 0967 strain was inoculated into 1 L (pH 7.0) and incubated in a small incubator at 28°C, 3 aeration/min, and 400 rpm.
The mixture was cultured with aeration and agitation for 120 hours.

The culture was centrifuged to obtain 3.07 L of the supernatant (containing 101,000 units of trehalostatin).
The pH of the solution was adjusted to 3.1 with hydrochloric acid, and the solution was then adsorbed onto a column (6 x 37 cm) of Dowex 50W x 4 (H) (Muromachi Chemical Co., Ltd.).
After washing with 3.5 L of 0.2 M pyridine-formic acid buffer (pH 3.2), the activity of inhibiting trehalase from Alder fly was assayed by adding 3.5 L of 0.4 M pyridine-acetate buffer (pH 4.2).
The solution was eluted with .

Active fraction (containing 120,000 units of trehalostatin)
The concentrate was concentrated to dryness under reduced pressure, the residue was dissolved in a small amount of water, and the pH of the solution was adjusted to 3.1 with hydrochloric acid. The insoluble matter in the solution was removed by centrifugation, and the resulting supernatant was poured into a Dowex 50W x 4 (H)
The column (3 x 12 cm) was loaded with 0.5 L of ethanol.
of deionized water, and then 3 L of 0.2 M pyridine-formate buffer (pH 3.2) containing 30% (v/v) methanol.
The target substance was eluted while washing with .

The target fraction (containing 120,000 units of trehalostatin) was concentrated under reduced pressure, and the residue was homogeneously suspended in 50% (v/v) acetonitrile and loaded onto a high-performance liquid chromatographic column (YMC-PA43, 2.5 x 27 cm). The column was washed with 65% (v/v) isocratic acetonitrile (flow rate: 5 ml/min). The eluate was fractionated in 5.0 ml aliquots while the eluate was monitored with a differential refractometer. The trehalase inhibitory active fraction (containing 119,000 units of trehalostatin) eluted at approximately 200 ml after loading.

The target fraction was concentrated and dissolved in 40% (v/v) acetonitrile, and loaded onto a high-performance liquid chromatographic column (TSK-Amide 80, 0.7 x 27 cm). The column was washed with a solvent of the same composition (flow rate 1 ml/min), and the eluate was fractionated in 0.5 ml increments while monitoring the eluate with a differential refractometer. The trehalase inhibitory activity fraction eluted at approximately 25-30 ml after loading. The target fraction (containing 118,000 units of trehalostatin) was concentrated to dryness, added with water, and lyophilized to yield 1 mg of the pure target product.

Example 2: Physicochemical properties of trehalostatin The substance isolated and purified from the culture of Amycolatopsis trehalostatica (SAM 0967) according to Example 1 was named trehalostatin.

The physical and chemical properties of this trehalostatin are shown below.

Properties: White powder.

Solubility: Soluble in water, slightly soluble in hexane, benzene, ether, and petroleum ether.

UV-visible absorption spectrum: No absorption maximum above 220 nm.

Color reaction: Positive Rydon-Smith reaction.

Ninhydrin reaction, 3,6-dinitrophthalic acid reaction, Elson
-Morgan reaction was negative.

Thin layer chromatography: Rf=0.37 Merck Kieselgel 60 F ₂₅₄ , developing solvent (n-butanol:acetic acid:water=3:1:2).

High performance liquid chromatography: Rt = 11.0 min. YMC PA03 (0.7 x 27 cm) Solvent: 65% v/v acetonitrile (in _H2O ) Flow rate: 1.0 ml/min Detection: differential refractometer.

Molecular weight: 366 (m/z 367, M+H, by SIMS) [α] _D ; +115° ^1H -NMR/ _D2O : 3.3 (ppm), dd, 1H 3.5 (ppm), m, 1H 3.5 (ppm), t, 1H 3.6 (ppm), d, 1H 3.6 (ppm), dd, 1H 3.7 (ppm), d, 1H 3.7 (ppm), dd, 1H 3.8 (ppm), ddd, 1H 4.1 (ppm), dd, 1H 4.2 (ppm), d, 1H 4.8 (ppm), ddd, 1H 5.2 (ppm), d, 1H ¹³ C-NMR/D ₂ O: 63.5 (ppm) 64.8 (ppm) 72.4 (ppm) 72.8 (ppm) 74.8 (ppm) 75.8 (ppm) 76.2 (ppm) 83.0 (ppm) 83.2 (ppm) 83.4 (ppm) 85.6 (ppm) 89.9 (ppm) 163.8 (ppm) The structural formulas estimated from the results below are as shown in Formula 1 and Formula 2 below.

Furthermore, the structure of trehalostatin was determined as follows. The chemical shifts for trehalostatin were 25
In the case of acetylated trehalostatin, the measurements were taken in deuterated water at 25°C, and in the case of deuterated water, the measurements were taken in _CDCl3 at 25°C. Unless otherwise specified, the chemical shifts in the text refer to the former.
DIS is an abbreviation for m-induced Differential isotope shift, and is defined as DIS = δ ¹³ CH ₂ O - δ ¹³ CD ₂ O (ppm). If a carbon atom has a deuterium-exchangeable functional group such as an -OH group or an -NH group bonded to it, the DIS of that carbon atom will have a positive value of about 0.02-0.1 ppm.

First, it was revealed that trehalostatin contains an α-D-glucopyranosyl group by the following MS, NMR, and chemical analyses: Liquid SIMS
The molecular ion peak of m/z 367 [M+H] ⁺ generated by Liquid Secondary Ion Mass Spectrometry (LIMS) was determined to be B/E-li.
Linked Scan (B/E constant link scan)
The presence of _a hexose residue was inferred from the formation of _{a fragment of [H-C6H10O5} ^] ₊ ^.
(Table B), and H, H-COSY spectrum (H/H
Homonuclear shift correlation NMR spectroscopy (Figure 1) revealed the spin system of the glucopyranosyl group, and furthermore, the coupling constant (J = 5.0 Hz) of the anomeric hydrogen (5.2 ppm) was determined.
Based on this, this was identified as an α-glucopyranosyl group. Furthermore, when trehalostatin was treated with hydrochloric acid-methanol, a monosaccharide was produced that exhibited the same mobility as methyl glucoside in silica gel thin-layer chromatography. This monosaccharide was acetylated and isolated by silica gel column chromatography. By comparing its ^1H -NMR spectrum with that of a standard, it was identified as methyl glucopyranoside (acetylated product), further confirming the presence of the glucoplanosyl group (Table A). This acetylated methyl glucopyranoside was converted to a tetraester of p-bromobenzoic acid and subjected to circular dichroism spectroscopy, revealing a peak at 244 nm.
Since the α-D-glucopyranosyl group gave a positive split CD signal, the absolute configuration of this glucopyranosyl group was determined to be D-type.
Anomeric carbon of glucopyranosyl group (83.4 ppm)
However, the upfield shift observed for the O-glucoside suggested that this α-D-glucopyranosyl group was an N-glucoside.

Next, the structure of the non-glucose portion of trehalostatin was determined as follows.

The degree of unsaturation of trehalostatin calculated from its molecular weight (366) is 4, and since this substance contains an α-D-glucopyranosyl group (degree of unsaturation 1), the degree of unsaturation of the non-glucose portion is 3. Furthermore, IR (1616 cm ^-1 ), UV (terminal absorption), and ^13C -NMR (163.8 ppm) suggest the presence of a ureido group (degree of unsaturation 1), so the non-glucose portion is considered to be bicyclic. ^1H -NMR, ^13C
-NMR, DEPT (Distortionless Enhancement by
The spectral data of each hydrogen and carbon of trehalostatin obtained by polarization transfer was analyzed using H, C-
The correlation was carried out by COSY spectroscopy (H/C heteronuclear shift correlation NMR spectroscopy) (Table B). In addition, each hydrogen and carbon of the non-glucose portion of the two isomers of acetylated trihalostatin was analyzed by HMQC ( ^1H -Detected Multiple Quantum
The correlation was performed using the Coherence Spectrum (Coherence Spectrum) (Figure 5).

The H,H-COSY spectrum revealed that the non-glucose portion contains a spin system with four hydrogen atoms (H3', H4', H5', and H6') at 4.2, 4.8, 4.1, and 3.8 ppm (Figure 1). The carbon atoms to which these hydrogen atoms are bonded (C3', C4', C5', and C6', respectively)
The magnitude of the chemical shifts of the acetylated trehalostatin (HMBC) and the acetylated trehalostatin ( ^HMBC ) of ...
ed Multiple-bond Heternuclear Multiple Quantum Coh
In the frequency spectrum, the hydrogen atoms at both ends of the spin system (H3', e.g., 5.92 ppm in the acetylated product; and H6', e.g., 6.35 ppm in the acetylated product) are
p ³ -quaternary carbon (C2', for example, 86.5 in the acetylated product)
From the cross peaks (Figure 2) for the sp 3 -quaternary carbon, it is clear that the above spin system forms a five-membered carbon skeleton together with this sp ³ -quaternary carbon. In addition, independent methylene hydrogens (H1'a, e.g., 3.94 ppm in the acetylated product; and H1'b, e.g., 1.00 ppm in the acetylated product) are also observed.
4.21 ppm) also gave a cross peak to the sp ³ -quaternary carbon (C2', for example, 86.5 ppm in the acetylated product) (Figure 2). From the above results, the following partial structure was suggested.

One of the terminal carbons (C3', 76.2 ppm) that make up the spin system described above resonates at a higher magnetic field due to acetylation (for example, 58.3 ppm).
5), it is suggested that a nitrogen atom is bonded to this carbon, and furthermore, this carbon has a DIS (>+0.0
7) was observed, indicating that an NH group is bonded to this carbon.
ppm methylene hydrogens (H1′a, H1′b) and 4.1,
The hydrogens (H5', H6') at 3.8 ppm shift significantly downfield (e.g., 3.9, 4.2,
5.4, 6.4 ppm (Figure 5)), and the carbons to which these hydrogens are bonded (C1', 63.5; C5', 83.2; C6',
83.0 ppm) were found to have hydroxyl groups bonded, which was confirmed by the DIS results (+0.06, +0.04, +0.07 ppm, respectively).
m), while 89.9 ppm of carbon (C4')
Since no DIS was observed (CIS<0) and acetylation did not result in a downfield shift of H4' (Table B and Figure 5), it was determined that the oxygen attached to this C4' was something other than a hydroxyl group. Based on these results, we hypothesized that the non-glucose moiety was a pseudocyclic sugar alcohol containing a ureido (isoureido) group (see Formula 3).

The relative configurations of the non-glucose moieties were determined by NOESY experiments as follows: 4.2 ppm (H3') and 4.
Between hydrogens (J = 8.6 Hz) at 8 ppm (H4') and 4.1 ppm
Between hydrogen (H5') and 3.8 ppm (H6') (J = 4.8 Hz)
Since NOEs were observed at each of these sites, it was found that they were in a syn relationship with each other.
Since there was no NOE between the hydroxyl group (H4') at 4.1 ppm and the hydrogen (H5') at 4.1 ppm (J = 2.7 Hz), it was found that they are in an anti-relationship (Figure 3). In addition, in the non-glucose portion of acetylated toehalostatin, an NOE was observed between the hydroxyl group attached to C2' (86.5 ppm in the acetylated product) and H3' (5.92 ppm in the acetylated product).
This shows that they are in a syn relationship (4th
In addition, of the two isomers of acetylated trehalostatin, one has an NOE between the methyl hydrogen of the N-acetyl group (2.66 ppm) and the hydrogen at the 3-position of the glucose residue (5.39 ppm in the acetylated product), indicating that this isomer has an acetyl group at the nitrogen atom bound to glucose, while the other is a positional isomer with an acetyl group at the N ^-3 ' position (Figs. 4 and 5).

From the above results, the structure of trehalostatin that satisfies the spectral data obtained was determined as follows (Formula 3): Note that the structure of the non-glucose portion may also be its enantiomer.

In order to inhibit trehalase, a ureido group,
N-α-glucopyranosides, in which one of the nitrogen atoms of the isoureido or amidine group is bonded, are thought to be effective.

Example 3. Enzyme inhibition spectrum of trehalostatin INDUSTRIAL APPLICABILITY The trehalostatin of the present invention exhibits an effective inhibitory effect on trehalase of insects, particularly blow flies, even at extremely low concentrations, and is useful as an insecticide against these insects.

───────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁵ Identification symbol Internal reference number FI Technical marking location (C12P 19/28 C12R 1:01)

Claims (7)

[Claims] Claim 1: A white powder that is soluble in water and sparingly soluble in hexane, benzene, ether, and petroleum ether, has an ultraviolet-visible absorption spectrum with no absorption maximum above 220 nm, is positive in the Rydon-Smith reaction, and is also positive in the ninhydrin reaction, 3,6-dinitrophthalic acid reaction, and Elson-Morg reaction.
The an reaction was negative, and the n-butanol:acetic acid:water ratio was 3:
The Rf was 0.37 in thin layer chromatography on Merck Kieselgel 60 F ₂₅₄ using a 1:2 developing solvent, and the flow rate was
The Rt was 11.0 min in high-performance liquid chromatography on a YMC PAO3 (0.7 x 27 cm) column using 65% v/v acetonitrile in _H2O at 1.0 ml/min, and the molecular weight was 366, [α]
_D : +115° and the following NMR spectra: ¹ H-NMR/D ₂ O: 3.3 (ppm), dd, 1H 3.5 (ppm), m, 1H 3.5 (ppm), t, 1H 3.6 (ppm), d, 1H 3.6 (ppm), dd, 1H 3.7 (ppm), d, 1H 3.7 (ppm), dd, 1H 3.8 (ppm), ddd, 1H 4.1 (ppm), dd, 1H 4.2 (ppm), d, 1H 4.8 (ppm), ddd, 1H 5.2 (ppm), d, 1H ¹³ C-NMR/D ₂ O: 63.5 (ppm), 64.8 (ppm), 72.4 (ppm), 72.8 (ppm). 74.8 (ppm) 75.8 (ppm) 76.2 (ppm) 83.0 (ppm) 83.2 (ppm) 83.4 (ppm) 85.6 (ppm) 89.9 (ppm) 163.8 (ppm) A new substance, trehalostatin, has the following values: 2. The trehalostatin according to claim 1, which is obtained by culturing an actinomycete belonging to the genus Amycolatopsis. [Claim 3] The actinomycete is Amycolatopsis trehalostatica S
3. The trehalostatin according to claim 2, which is AM 0967 (FERMP-10544; BP-2784). 4. A method for producing trehalostatin, which comprises culturing the actinomycete belonging to the genus Amycolatopsis that produces the trehalostatin according to claim 1. [Claim 5] The actinomycete is Amycolatopsis trehalostatica S
5. The method according to claim 4, characterized in that the strain is AM 0967 (FERMP-10544; BP-2784). Claim 6: Amycolatopsis trehalostatica Am
ycolatopsis trehalostatica SAM 0967(FERM P-10544;
BP-2784). 7. Amycolatopsis trehalostatica S. containing the trehalostatin according to claim 1, which exhibits an inhibitory effect on trehalase of insects, particularly blow flies.
Culture of AM 0967 (FERM P-10544; BP-2784).