JPS5878592A - Amylase inhibitor ki-26a and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel amylase inhibitor KI-26A, by cultivating a microorganism belonging to the genus Streptomyces. CONSTITUTION:A microcoganism, e.g. Streptomyces griseofuscus var. TA-26 (FERM-P No. 6171), belonging to the genus Streptomyces, and having the ablility to produce amylase inhibitor KI-26A is inoculated into a liquid nutrient culture medium, and cultivated at 5.0-8.0pH and 20-40 deg.C under aerobic conditions for 1-6 days to collect the aimed amylase inhibitor KI-26A mainly from the culture fluid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the invention.
The present invention relates to a novel amylase inhibitor KI-26 which can be obtained by culturing Scus variety T mu-26, and a method for producing the same.

In recent years, amylase inhibitors have attracted attention in various directions, and the following methods are being considered as ways to utilize them.

(1) Medications for the Treatment of Diabetes and Obesity The treatment of diabetes and obesity is mainly based on dietary therapy, which inevitably results in pain for the patient. Therefore, if you take amylase inhibitors when eating starchy foods such as rice and udon, which are the mainstay of the Japanese diet, the breakdown of starch into carbohydrates such as glucose will be inhibited. You can expect it. As a result, it is thought to be useful in curing urinary tract disease and obesity.

(2) As a diagnostic agent for pancreatic disease, it is known that when a pancreatic disease occurs, the degree of pancreatic cleavage aryase chain in the blood increases. Therefore, it is possible to separately quantify only the amount of pancreatic amylase in the blood, distinguishing it from the salivary amylase. In other words, if we use an inhibitor that has a 1r1 inhibitory activity against either amylase, pancreatic amylase,
Since the amount of amylase enzyme in each saliva source can be accurately fixed, a more reliable diagnosis of pancreatic disease can be made.

In this way, Amitsu-1! F inhibitors are being considered for use in various fields and are expected to be of high value [
There is.

As a result of intensive research into microorganisms that produce amylase inhibitors, the present inventors have discovered a substance having amylase inhibitory activity in a culture of a specific microorganism belonging to the genus Stregutomyces, and have completed the present invention. reached.

That is, an object of the present invention is to provide a novel amylase inhibitor KI'-2<SA. As the producing bacteria of KI-26mu used in this project #1,
Streptomyces griseofuscus variety TA-26 (1ltr@p) belonging to the genus Streptolyces
ts+Fe*a grimsefwcmsmasr,
TA-26) was used, and this strain was newly isolated from the soil of Ilwa City, Saitama Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Chokoken-Ki No. 6171. It exhibits the following amphibious characteristics.

1) Morphology Sporulation 1 The branches of the filament are simple and loop-shaped. The spores have a number of 10 or more, and the surface structure of the spores is smooth when observed with an electronic microscope. Spore size ranges from Q,9 to tIXt1 to t3.

No sacs were observed, and the epiphyte of the turtle spore stalk was on the aerial hyphae.

) Growth status in each medium Shown in Table 1.

Physiological properties of Myanmar (1) Growth temperature range: 206-40℃, optimum growth temperature is 60℃
It is.

(2) Liquefaction of gelatin Slight liquefaction is observed.

'(3) Hydrolysis of starch Hydrolysis of starch is observed.

(4) # of skimmed milk! Solid peptonization Condensation and peptonization are observed.

(5) Generation of melanin-like pigments No pigment generation
After inoculating the bacteria, the cells were cultured at 30°C for 2jI to examine the assimilation ability of the carbon source, and the following results were obtained.

L-arabinose, D-xylose, D-/lucose,
D-7 lactose, 1)-Ynnit, D-galactose, and suricin are assimilated, but L-rhamnose, p-sucrose, raffinose, and inositol are not assimilated.

As mentioned above, B@rg@
ya manualof D*t@rminativ*
Bact@riology 8th edition 1974, Ist
*rmatl@mal Journal of 5
yst@maticBacteriol@gy Vol.
16.45 1966, Vo'118. A2 196
8, Vel 22.44 1972.

B@rg@y's manual of D@t@rm
1mativeBa@t*rlogy 8th edition 197
According to the 4th year, the cocoon color of this strain is Qray;
It falls into minute II of 17.42 f because the moist shape of the air # branches is spiral, no melanoid pigment is produced, and the surface of the spore is smooth. Table 2 shows a comparative study of the mycological properties of strains that fall into these categories and are similar to the present strain, as well as strains for which amylase inhibitor production layers are known.

Streptomyces diastateicus (8t, diastatii) is known to have the ability to produce amylase inhibitors.
eus), Streptomyces tendera (8t, t
@nda・), Streptomyces a and leofascens (8t, aur@ofaei・n■), Streptomyces myelinus (Stomarinlls), etc., differ from this strain in terms of the color of living hyphae and ability to assimilate carbon sources. [There is. Streptomyces diastatius (8t, diastatius) is characterized by the fact that the color of the living hyphae is gray-yellow or yellow-green, that it has the ability to assimilate cisucrose, and that the colony morphology on yeast malt agar medium, etc. It is clearly different from this mycelium because of the different things. Streptomyces tenda (at, t/n
The color of the viable hyphae of da.) is yellow or yellow-green, and the ability to assimilate rhamnose, sucrose, and inosite differs from this strain. Streptomyces araleofaciens (8t, aur@ofaisms) is sucrose. Streptomyces murinas (8t0mri-1111111) differs from this strain in that the color of its single living hyphae is gray-yellow, and that it does not have the ability to assimilate arabinose and hydrolyze starch.

Streptomyces argenteolus (at, arg
＠nt＠o1ms) is similar to this strain in terms of the color of the bacterial flora and the color of single living hyphae, but there are differences in the ability to assimilate rhamnose and the concentration of NaCl weight resistance. It is different from this strain.

Streptomyces griseofuscus (8t, gr
im@-・halle11g) is very similar to this strain in terms of the color of the bacterial flora and the ability to assimilate the carbon source of one living hyphae.

However, there is a problem in identifying this strain as the same species because there are differences in the degree of growth on Sheavec medium and Streptomyces griseofuscus has no ability to produce amylase inhibitors.

However, since no strong mycological characteristics were observed to determine this strain as a new species, the present inventors identified this strain as a member of the genus Streptomyces and named it Streptomyces griseophuscus var. TA-26. KI-26A, the target substance of the present invention, is produced by a conventional method under aerobic conditions in a liquid medium containing known nutrient sources such as carbon sources, nitrogen sources, and inorganic components that can be used by ordinary microorganisms. Obtained by culturing. Carbon sources include starch, polysaccharides such as amylose, amylopectin, oatmeal,
Use molasses or a mixture of these. The concentration used is preferably 0.5 to 10 ml. As the nitrogen source, protein, protein hydrolyzate, amino acid, peptone, casein hydrolyzate, cornstarch liquor, soybean flour, meat extract, yeast extract, etc., or a mixture thereof can be used. Inorganic components include magnesium, potassium, sodium, iron, and zinc.

Phosphates, sulfates, and chlorides such as manone are used, and the concentration used is 1 to 4.0% by weight.

Further, in order to suppress foaming during fermentation, 10% by weight or less of an antifoaming agent may be added. As the antifoaming agent, common antifoaming agents such as silicone and soybean oil are used.

As for the culture method, aerobic liquid culture such as shaking culture and aerated culture is suitable, and the pH
Cultivate for 6 days, preferably at pH 6.5 to 5, and for about 3 days at 30°C to 37°C.

In addition, in order to produce KI-26A at a high yield, K is added using an appropriate control method to keep the specific growth rate of bacterial cells constant, rather than adding a carbon source all at once and culturing in 5 batches. Condition i control, i.e. semi-batch culture,
For the production of KI-26, it is desirable to control the growth rate of bacterial cells using a known culture control method such as continuous culture. The specific growth rate of Soda to be controlled is αo2~a1
It is desirable for leprosy in KI-26 to maintain the temperature between O,OS and α1 hr-' for 5 hr-1.

In the culture method described above, KI-26 is easily produced in the culture fluid.

A possible method for measuring amylase inhibitory activity is to measure the amount of decrease in amylase activity. Methods for measuring the enzymatic activity of Aden2-ase include: 1) A method based on the amount of reducing sugar produced by starch decomposition (D
NS method, etc.) It) A method of observing the amount of starch reduction in the W starch reaction (
The blue price method) is known.

The DNS method (Satoshi Fukui, Quantitative Method for Reducing Sugars, University of Tokyo Press, 1969) utilizes a reaction in which a DNS reagent reacts with reducing sugars and turns brown; due to the increase in reducing sugars, Measure enzyme activity. The measurement method is sample α511
1JKfl elementary solution (e.g. porcine pancreatic α-amylase,
O.D. Add α5d (dissolve near α1 in αIM, pH 6,9, phosphate buffer) and heat at 67°C.
Let it react for 0 minutes. Furthermore, 1% starch solution (αIM, pH
49, dissolved in phosphate buffer) 1d, and then 5
React for 10 minutes at 7°C. Next (, DNB reagent 511
1 to stop the reaction. This solution is further boiled for 5 minutes, 15alJ of water is added, and the absorbance at 550fi is measured.

As a control, add sample α5ajK# elementary solution [1L5-, react for 10 minutes, add 3 d of DNS reagent, and further add 1 d of 1% starch solution to keep the reducing sugar concentration constant. Enzyme activity can be measured from this change in reducing sugar concentration.

The blue price method uses sample α58/[1% starch solution (11M, p
Add H1h9, phosphate buffer) 1-, and further add 0.5- of the enzyme solution, and react at 37°C. After reacting for 10 minutes, α2- was released and boiled for 3 minutes.

Then add 1/3000 normal iodine solution of 5IIj and 6
Measure the absorbance of 60011. As a control, an enzyme solution inactivated by heating is used.

In addition, in the case of the DNS method, the definition of amylase activity is that the enzyme titer that releases α1 kg of glucose per minute is 1 unit, and in the case of the blue price method, the activity per 1 d of enzyme solution is calculated from the following formula. do.

(OD) m: Absorbance (OD) of a control reaction solution using water instead of enzyme solution m: #Absorbance of elementary reaction Amyl (The unit of inhibitory activity is the amount of inhibitory substance that inhibits 50% of 1 unit of amylase activity. To isolate the substance KI-26A produced by the present invention (with an activity of 15 units),
For example, do the following 5. After culturing the bacteria, add 1 dose of activated charcoal to the supernatant after removing the bacteria from the culture solution by centrifugation.
% (W/V) to adsorb KI-26A onto activated carbon. Thereafter, the activated carbon is separated and washed with water, and the amylase inhibitor is eluted using an organic solvent such as ethanol, methanol, or acetone.

This eluate was condensed under reduced pressure to form a concentrated solution OW@X 5
It is adsorbed onto an ion exchange resin such as QWX2 (trade name, manufactured by Dow Chemical Company) and Amberlite IR-120 (H+Il) (trade name, manufactured by Roll & Hart Company). Thereafter, it is washed with water, and elution is performed with 1N aqueous solution, etc., to elute the A-kP inhibitor. After compressing the eluate under reduced pressure, it was filtered through gel e-filtration (trade name: SE7ADEX G-25,
KI-26A can be isolated and purified in accordance with a conventional method by appropriately combining a method such as Pharmacia Co., Ltd., Toyo Pearl HW-50 (trade name, Toyo 1Datsu ■ Co., Ltd.), cellulose column chromatography, etc. KI-2 separated after 5K
The sample is freeze-dried to obtain a white powder sample.

Amylase inhibitor KI-2t obtained after 5K
The physical and chemical properties of SA are as follows.

(a) Elemental analysis: carbon 4118%, hydrogen 6.58%, nitrogen 198%, oxygen 5126% (b) Molecular weight When LI filtration is performed using Toyopearl HW-50, KI-26 has a molecular weight of 2200. It eluted at position ~2500.

Figure 1 shows the results of all determination using a potassium bromide disk.
850°7(So, 700,580,520 (d)H-NMR spectrum measurement results are shown in FIG. 4.

'#pm: +215, 550. 572, 5.
The results of 50(·)''C-NMR spectrum measurement are shown in FIG.

J PPm: 19.5, 42.7, y5,7
9.1.10t7(f) Browning begins at melting point 199°C and carbonizes at 218°C.

(g) Solubility Soluble in water. Insoluble in acetone, methanol, ethanol, chloroform.

(h) Color reaction.

Negative for Anthrone sulfuric acid reaction, Somogyi Nelson reaction, phenol sulfuric acid reaction, and positive ninhydrin reaction.

(1) Appearance white powder (j) UV spectrum No KlI absorption in the ultraviolet region.

(k) Components An oligosaccharide whose main component is glucose.

(1) Enzyme inhibitory activity The inhibitory activities of KI-26A and its decomposed substances against various enzymes were investigated.

The amylase inhibitory activity of +11 K1-26 was measured according to the two methods described above. The target amylase is saliva amylase, B, aubtilu liquefied amylase originating from Hatake, and Rhizopus nippeus (Rh1zopus nippeus).
These include glucoamylase originating from corn and sweet potato β-amylase.

For the measurement, the concentration of KI-26m was set at 10 μC during the enzyme reaction, and those with amylase inhibitory activity of 50% or more were selected as (-
+ and less than that are represented by (→).The result is shown in the 6th
Shown in the table.

(2) Enzyme inhibitory activity of KI-26A degrading substances It has been reported that a substance with amylase inhibitory activity decreases its activity and newly increases its inhibitory activity against sucrase and others through acid decomposition. (From@r
.

W,, Naturviss@nmehaft@n
64. 535 (1977)) [Hiroshi Hara, Shinobu Namiki, Japanese Society of Agricultural Chemistry Lecture Abstracts, 1980, P9] Therefore, KI-
The presence or absence of inhibitory activity against sucrase and α-glucosidase was investigated for the 26A degrading substance. That is, KI-
Dissolve 26 μm 50 da in 6% H, 8042111, 10
React at around 0°C for 4 hours. After the reaction, neutralize with 1N sodium hydroxide and use as a sample for inhibitory activity measurement.

First, as a method for measuring the inhibitory activity of sucrose, the enzyme activity can be measured by quantifying the glucose produced by the enzymatic decomposition of sucrose into 7-lactose and glucose using an enzymatic glucose measurement method.The specific method is as follows: , α05M acetate buffer (pt'14.5)2
Add 1 d of 1% sucrose solution to 111. For the reaction, 1 ml of an enzyme solution α of an appropriate concentration is added and the reaction is carried out at 25°C.

After starting the reaction, the reaction is stopped by heating over time, and the glucose concentration is measured after cooling with water. Enzyme inhibitory activity can be determined by adding the sample to the above reaction system and comparing the glucose concentration with a control sample.

Next, as a method for measuring the inhibitory activity of α-glucosidase, α1M potassium phosphate buffer (pH 7,0) 5
Add 1d1 of reduced glutathione solution (1w/j) to 1'1
Further, to 1, add a solution of phenyl-α-D-curcoside (SMg/a/). To start the reaction, α1d, an enzyme lacquer solution (manufactured by Sigma, originating from baker's yeast), is added and the reaction is carried out at 37°C. For the measurement of inhibitory activity,
# Add an appropriate concentration of inhibitory solution α1d before addition. After starting the reaction, the reaction is stopped by heating over time, and after cooling with water, the absorbance at 400 η is measured. The inhibitory activity can be determined by measuring this change in absorbance.

When the inhibitory activity against the two enzymes was measured in this way, no inhibitory activity against sucrase and α-glucosidase was observed in the acid hydrolyzate of KI-2 (5).

The amylase inhibitor KI-26A used in the present invention is as described above, but the following amylase inhibitors are currently known as amylase inhibitors produced by microorganisms, and the differences with these substances have been compared and described below. show.

(a) Nojirimycin of Tomizo Niwa et al. [Agr, Biol, Chem, 54, 966
(1970) ) Since nojirimycin is a monosaccharide annoic acid, it is different from KI-26A of the present invention, which is an oligosaccharide.

Stem) Seinosuke Ueda et al.'s peptide-like substances [Mugr, Blot, Chew, 57.202O25
(1975)) (A, Blot, Chew, 40.11
67 (1976)) The present invention differs in that it contains amino acids as a constituent! It is different from 26mm.

(c) Oligosaccharides mainly composed of glucose, such as Murakite Sawao et al.
[JP-A-50-123891] [JP-A-55-71494] Adsorption of amylostatin to ion exchange resins has not been observed. Also, amylostatin B, ClD%E%
F is a substance with a molecular weight of 600 to 1550, and has a different molecular weight from the present substance KI-26. Furthermore, amylostatin E and F have different properties from KI-26, such as having an inhibitory effect on bacterial liquefaction amylase.

) Amylase inhibitor by Kiyoshiki Ueda et al. [JP-A-51-54990] The molecular weight of this amylase inhibitor is about 600, and it is KI.
-Different from the molecular weight of 26 μm.

(e) TAI-A%TAI-8 by Shinbe Namiki et al. [JP-A-54-106402] [JP-A-54-106403] TAI-A and TAI-B contain a methyl group in the molecule. However, KI-26 has been found to contain a methyl group based on NMR spectroscopy, and differs from TAI-A and TAI-8 in this respect.

(f) Trestatin compounds by Teru Yokose et al.
-163511] The trestatin compound has a molecular weight different from that of KI-26, and has a molecular weight of 2.4 to 2.2 mm in the H-NMR spectrum.
Absorption was observed around 5 ppm, whereas KI-26
There is no absorption in the vicinity. Furthermore, all trestatin compounds contain trestatin in their molecules, and are different from KI-26A in this respect as well.

()) W, Fromm@r et al.'s amino sugar compounds (Naturwlas*n5ehaft@n 64,
535 (1977)) These substances are amino sugar derivatives represented by the following molecular formula.

These are oligosaccharides mainly composed of glucose, and have many similarities with KI-26A, such as containing a methyl group. Inhibitory activity was observed, whereas KI-
26A shows no sucrase inhibitory activity due to acid hydrolysis.In what respects is the substance KI of the present invention
-26A is a different substance.

As is clear from the above, KI-26A is a new substance that strongly inhibits amylase activity.

Next, embodiments of the present invention will be described.

Example: Streptomyces griseofuscus variety TA-26 (Feiko Kenzonoyo No. 61
4 tscc of sterile water was added to the slant on which No. 71) was grown to create a suspension. Add 1 cc of this suspension to starch 511
/I, glucose 511/I, peptone 11i/j, yeast extract 111/1. Potassium phosphate 11I/l, disodium phosphate 111/I, magnesium sulfate α51
A 500 d capacity Sakaro flask containing 100 aj of a 1/l, pH 7,3 liquid medium was inoculated. The culture temperature is
The cells were cultured at 30° C. for 2 days, and this culture solution was used for culture using 1 ol of jar fermenter (Customer Attractor 51). The culture conditions were a temperature of 30°C, a pH of 7, and a stirring speed of 40.
The test was carried out at 0 rpm and an air flow rate of 1 mm. The amount of inoculation is 2%
The same medium components as above were used. After culturing for 6 days, the bacterial cells were centrifuged and the bacterial cells were removed.
Obtained. The inhibitory activity of this f solution was 2700 units/d. Add 1% (W/V) of activated carbon to this rllllEK.
After adsorbing I-26, the activated carbon was separated, washed with water, and eluted with 90% ethanol containing 4% acetone.

After concentrating the eluate under reduced pressure, DEW@! 50WX2 (50
~100 mesh, H type → adsorbed on ion exchange resin.

After washing with water, eluate with 1N ammonia water, concentrate, and gel filtration (Toyopearl HW-50. Column 24 × 50
The fraction with inhibitory activity was freeze-dried to obtain KI-26A in the form of a white powder.
30011F was obtained.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of amylase inhibitor KI-2SA. Figure 2 shows the amylase inhibitor KI-26) l-NM.
The R spectrum is shown. Figure 5 Huff Miller 4'ffi Hazardous Substance K I-26
AIF)”C-NMR spectrum is shown. Patent applicant: Nihon Inchu Kagaku Kogyo Co., Ltd.
Kawamura Physical and Chemical Research Institute

Claims (1)

[Claims] L: Amylase inhibitor KI-26 having the following properties:
A(a) Elemental analysis value 41.181! , water gas, nitrogen α98-1 acid volume t2SIG (1+) molecular weight 2200-2500 (e) rR spectrum shown in #11 (d) H-NMR spectrum shown in Figure 2 (,) IIC-) Ji ( R, (g) Solubility Soluble in water, acetone, methanol, ethanol, and tetraroform. (h color reaction Anthrone sulfuric acid reaction, Somogyi-Nelson reaction, phenol-
Amylase inhibitor K belonging to the genus Streptomyces.
amylase inhibitor KI-2+6A, which is characterized by culturing microorganisms capable of producing amylase inhibitor KI-26A, accumulating amylase inhibitor KI-26A in the culture solution, and collecting the amylase inhibitor KI-26A. Production method: The production method according to claim 2, wherein L is a microorganism or Streptomyces griseofuscus varietate-26.