KR900007643B1 - Novel microorganism streptomyces sp.y-125 - Google Patents

Abstract

Streptomyces sp. Y-125 for producing anylase inhibitor (I) is screened from soil. The screened strain <KCTC 8387P, ATCC 53890> is cultured in the medium contg. yeast extract, soy protein, peptone, soluble starch, inorganic salt e.g. NaCl and antiforming agent at pH 6.0-7.0, 25-30 deg.C and aerobic condition for 2-4 days., and the (I) is obtained by centrifuging (30000-40000 rpm) the culture broth, concentrating the centrifuged soln. at 50-70 deg.C under pressure, etc.. The optical pH and M.W. of the obtained (I) are each 2-12 and 700-1500.

Description

Novel microbial Streptomyces sp.Y-125 and amylase inhibitors prepared therefrom

1 is a graph showing the stability of the amylase inhibitor purified according to the present invention.

2 is a graph showing the thermal stability of the amylase inhibitor.

3 is a diagram showing an infrared spectrum of the amylase inhibitor.

The present invention relates to a novel microorganism Streptomyces sp. Y-125 and amylase inhibitor prepared therefrom.

In general, carbohydrates, one of the three major nutrients required by the human body, are consumed in the form of disaccharides or polysaccharides, and then glycoside hydrolase enzymes such as amylase, maltese or saccharace are absorbed just before being absorbed into the body. ) Decomposed by the action of the bar, the excess sugar is produced in the body, diabetes, obesity, hyperlipidemia, gastritis, stomach ulcers, duodenal ulcers and the like.

Therefore, in order to prevent and treat these diseases, a method of inhibiting the production of excess sugar by inhibiting the action of various glycolytic enzymes in the body by using inhibitors of glycolytic enzymes is known (USP No. 4,307,194, 4,451,455), and these glycolytic inhibitors are known to be produced abundantly in actinomycetes, especially Streptomyces.

Conventional glycolytic enzymes generally have high inhibitory effects on amylases at high molecular weights, while low molecular weights have been reported to have excellent inhibitory effects on maltease or saccharace, but the present invention is relatively The present invention relates to an amylase inhibitor having a low molecular weight and specific inhibitory effect against amylase.

Conventionally known methods for producing amylase inhibitors are chemical methods in addition to biological methods, ie, small molecules such as salicylic acid or abiscisn and non-specifically inhibiting enzymes or enzymes by physical adsorption. It was prepared by using a polymer material to denature and precipitate.

However, these conventional amylase inhibitors only affected salivary amylase and had little effect on pancreatic amylase (E. KNEEN, RMST-ANDSTEDT, ARCH., BIOC.BIOPHY, 9, P 235). 1946). Other amylases have been reported to have relatively low activity because they are nonspecific but hardly inhibiting, as well as being heat-stable and inactivated by trypsin (USP 4,282,318).

Accordingly, the present inventors have discovered a novel microorganism having high activity in producing an amylase inhibitor, and continued extensive research on the isolation and use thereof, and as a result, the amylase inhibitor produced by the new microorganism was compared with the conventional amylase inhibitor. The present invention is made on the basis that the activity and stability are much better.

An object of the present invention is a novel strain producing an amylase inhibitor and a method for effectively producing a high yield amylase inhibitor using the same. And to provide an amylase inhibitor prepared therefrom.

Hereinafter, the present invention will be described in detail.

Streptomyces sp.Y-125, a microorganism strain according to the present invention, is a new strain isolated and cultured from soil samples collected from Chuncheon, Gangwon-do and Osan, Gyeonggi-do, Korea. It was deposited with the Institute of Science and Technology under accession number KCTC 8387 P and on March 31, 1989 with accession number ATCC 53890.

The following is a step-by-step description of the phylogenetic characteristics of the new strain Streptomyces sp.Y-125. MANUAL OF DETERMINATIVE BACTERIOLOGY, Vo1.2, 1986).

1) Cultural Characteristics

Streptomyces sp.Y-125, a new strain according to the present invention, produces abundant continuous mycelium belonging to the gray (GY) series of spores. Such culture characteristics are shown in Table 1 of Oatmeal agar (ISP). No. 3), inorganic salt starch agar (ISP No. 4) and tyrosine agar (ISP No. 7), especially in oatmeal agar (ISP No. 3), the growth state and color are clearly observed. The color on the back is yellow-brown, which is not affected by pH. Yellow pigment is produced in tyrosine agar medium, and pale yellow pigment is produced in skim milk medium, and no soluble pigment is produced on other medium.

TABLE 1

1 / ISP = International Streptomyces Project Badge

(International Streptomyces Project Media)

2 / SM = Skim Milk Agar Media

3 / NT = Nutrient Agar Media

4 / GA = glucose-asparagine agar plate

(Glucose-Asparagine Agar Media)

2) morphological characteristics

The morphological characteristics of Streptomyces sp.Y-125 grown on oatmeal agar produce a branched mycelium that does not divide and does not form spores.

In addition, spores form at the end of the mycelia, and the mycelia are wavy, and spores are spherical or rod-shaped. The surface of the spores is smooth and has a size of 0.7-1.2 × 1.2-l.8 μm.

At this time, the morphological characteristics were examined by light microscopy, and the surface and size of the spores were observed by scanning electron microscopy.

3) Physiological characteristics

Table 2 shows the carbon utilization form of Streptomyces sp. Y-125, and the carbon utilization was measured using an ISP medium having a final concentration of 1.0% by adding a sterile carbon source.

At this time, the incubation temperature is 30 ℃, the result was measured after 14 days.

TABLE 2

-: Not available +: Used

On the other hand, Streptomyces sp. Y-125 hydrolyzed starch, while skimmed milk and gelatin did not hydrolyze, neither reduced nitrate to nitrite nor fiber.

In addition, these strains proliferated at 20-80 ° C., particularly at 25-30 ° C., proliferating and producing melanin pigment in aerobic and tyrosine agar (ISP No. 7).

As described above, Streptomyces sp.Y-125 according to the present invention has been separated into novel strains because there are various differences from known species when comparing the morphological, culturing and physiological characteristics of the known species ( Note: BERGEY'S MANUAL OF DETERMlNATIVE BACTERIOLOGY, Vo1.2, p1383-1418, 1986).

Meanwhile, the amylase inhibitor produced by the new strain Streptomyces sp.Y-l25 according to the present invention is composed of polysaccharides and amino acids, and its activity is very high at 12,000 AIU / mg, and is boiled for 120 minutes. It also appeared to have no effect on the inhibitory properties.

At this time, 1 unit of amylase inhibitor (1 ATU) is defined as the amount of inhibitor when the unit of amylase is inhibited by 50%, and the amylase unit is defined as the amount of appeal when 1 μm of glucose is produced in starch for 1 minute. The resulting glucose was measured by reducing sugar with 3,5-dinitrosalicylic acid and a standard curve was selected as maltose.

Meanwhile, in the present invention, amylase inhibitor activity was measured by the following method, that is, 2 × 10 ⁻³ % amylase solution (10−) in 100 mM Tris-HCl buffer solution (pH 7.0) containing 5 mM calcium chloride. 20 AIU / ml) 0.5 ml is dissolved, and 0.5 ml of inhibitor solution (0.300 Ug) or culture solution is added thereto, followed by mixing for 10 minutes at 37 ° C., and 1.5% solubility in 100 mM Tris-HCl buffer solution. 2 ml of starch solution was added and reacted for 10 minutes.

Subsequently, 2 ml of 3,5-dinitrosalicylic acid was added, heated for 5 minutes, cooled sufficiently, diluted 10-fold with distilled water, and absorbance was measured at 546 nm.

On the other hand, the control test was carried out in the same manner as above except adding 0.5ml of distilled water instead of the inhibitor solution, and the blank test was added 0.5ml of 10 mM Tris-HCl buffer (pH 7.0) instead of amylase solution, 0.5ml of distilled water instead of the inhibitor solution. It was carried out in the same manner as above.

T.C and B are the absorbances of inhibitor test, control test and blank test respectively.

On the other hand, the amylase inhibitor according to the present invention was also very stable to pH and stable at pH 2.0 to 12.0, and therefore, it is expected that its activity will not be reduced during the degradation route.

In addition, the molecular weight measured by Sephadex G-25 is 700 to 1500, and despite the relatively small molecular weight, it exhibits sufficient inhibitory effect against amylase, maltese and saccharace, especially pancreatic amylase rather than saliva amylase. The inhibitory effect on was found to be more excellent.

Hereinafter, a method of culturing the novel strain Streptomyces sp. Y-125 and a method of separating the amylase inhibitor from the culture are described in more detail as follows.

(I) Strain Culture

Dilute hydrochloric acid solution is added to a culture medium containing yeast extract, skim soybeans, nitrogen sources such as peptone, soluble starch, carbon sources such as glucose, and inorganic salts such as sodium chloride, and the pH is adjusted to 6.0 to 7.0, and the pH is adjusted to 6.0 to 7.0 for 15 minutes. After sterilization, bacteria were inoculated and incubated for 2-4 days at 25-30 ° C. under aerobic conditions.

The following Table 3 shows the activity of the inhibitor for various carbon sources (concentration 1%), Table 4 shows the activity of the inhibitor against the nitrogen source, as shown in Table 3 and Table 4, the carbon source is soluble starch, nitrogen source is The highest activity was found in the mixture of yeast extract and polypeptone.

In this case, an antifoaming agent may be used, and the antifoaming agent is generally useful for preventing excessive foaming, and a conventional antifoaming agent such as a silicone antifoaming agent is used.

TABLE 3

At this time, the basic medium composition is 0.5% juicy, 0.5% polypeptone, 0.3% sodium chloride, the pH of the medium is 7.0.

TABLE 4

At this time, the basic medium composition is 1% soluble starch, 0.3% sodium chloride, 0.05% magnesium sulfide, 0.1% calcium phosphate, 0.001% copper sulfate, the pH of the medium is 7.0.

(II) Isolation of Amylase Inhibitors

Separation of the culture medium and the bacteria from the strain culture can be carried out by conventional methods such as centrifugation or filtration using a filter aid such as Celite. Thus, the amylase inhibitor is separated from the culture medium. In order to separate, it can be separated by a general method, that is, such as lyophilization of the culture solution, salting out, or organic solvent precipitation, adsorption, or ion exchange resins, such as gel filtrate. .

A) The strain culture was centrifuged (30,000-40,000rpm) to separate the cells and the culture medium, and the culture solution was concentrated to 1 / 5-1 / 10 under 50-70 ° C reduced pressure (10-50mHg).

The precipitate is then filtered off and the concentrate is lyophilized if necessary.

B) Precipitate the inhibitor by treating the centrifuged culture or concentrate with a hydrophilic solvent such as organic solvent, methanol, ethanol or acetone.

Since impurities are precipitated at low concentration, it is easy to remove impurities at around 60-70%.

C) Precipitate the precipitate using salts such as ammonium sulfate or sodium chloride.

The precipitate is dialyzed or washed directly with an organic solvent for dialysis and drying.

D) Adsorption by ion exchange resin

This process is the most suitable method for separating amylose inhibitors when they have polarity. The inhibitors are eluted by changes in ionic strength or pH and separated through gel filtration using molecular weight.

In addition to the above separation methods, precipitation of impurities by thermal denaturation, permeation of molecular membranes by molecular weight, ultrafiltration, and the like may be used.

Hereinafter, examples of the present invention will be described.

Example 1

진탕 플라스크에다, 0.1% 가용성 전분, 1% 포도당 ,0.5% 육즙, 0.5% 펩톤, 0.3% 염화나트륨으로 이루어진 pH 7.0의 배지 400ml를 넣고, 121℃에서 15분간 살균후, 미리 배양한 스트렙토 마이세스 sp.Y-125 균현탁액 20ml를 접종하여 4일간 배양하였다.2

In a shake flask, 400 ml of a medium of pH 7.0 consisting of 0.1% soluble starch, 1% glucose, 0.5% gravy, 0.5% peptone and 0.3% sodium chloride was added, sterilized at 121 ° C. for 15 minutes, and then pre-cultured Streptomyces sp. 20 ml of Y-125 bacteria suspension was inoculated and incubated for 4 days.

After culturing, the cells and the culture medium were separated by centrifugation, and 270ml of 15AIU / ml of the culture solution was concentrated at 40-60 ° C. and reduced pressure (10-50mHg) to obtain a 30ml concentrate.

Ethanol 90% was added to the concentrate, and the precipitate thus formed was separated by centrifugation. The precipitate was dissolved in distilled water and lyophilized for 36 hours to obtain 0.07 g of amylase inhibitor, which is 2 × 10 ⁴ AIU / g.

Example 2

5 ml of 2 L shake flasks, each containing 400 ml of a medium of pH 7.0 consisting of 1% soluble starch, 0.5% peptone, 0.5% broth and 0.3% sodium chloride, sterilized at 121 ° C. for 15 minutes, and then incubated with pre-cultured bacteria 20 ml of sp. Y-125 was inoculated and incubated at 28 ° C. for 4 days.

Subsequently, the cells and the culture were centrifuged to obtain 1500 ml of 85AIU / ml of the culture solution, and the culture solution was concentrated and filtered to 150 ml under reduced pressure of 50-60 ° C. to remove precipitate impurities, and 60% of the ethanol was removed to remove the precipitate and the filtrate was concentrated. .

The concentrate was lyophilized for 24 hours to obtain 6.4 g of amylase decontaminate, 3 × 10 ⁵ AIU / g.

Example 3

진탕 플라스크 10개의 실시예 2와 같이 배양하여 배양액 3000ml를 얻고, 이 배양액을 60-70℃감압하에서 1/10로 농축한 후, 에탄올 90%로 처리하여 그 침전물을 게거하고, 여액을 다시 농축하여 얻어진 농축물을 증류수에 용해한 다음, 24시간 동결 건조시켜 4.7×10⁵AIU/g인 10.2g의 아밀레이스 저해제를 얻었다.2

10 ml of shake flasks were cultured in the same manner as in Example 2 to obtain 3000 ml of the culture solution, and the culture solution was concentrated to 1/10 under reduced pressure of 60-70 ° C., treated with 90% of ethanol to remove the precipitate, and the filtrate was concentrated again. The obtained concentrate was dissolved in distilled water, and then lyophilized for 24 hours to obtain 10.2 g of amylase inhibitor which was 4.7 × 10 ⁵ AIU / g.

Example 4

진탕 플라스크 5개에다, 2% 가용성전분, 0.5% 폴리펩톤, 0.5% 육즙, 0.3%염화나트륨으로 이루어진 pH 7.0의 배지를 각각 400ml씩 넣고, 이를 121℃, 15분간 살균한 후, 균액 스트렙토 마이세스 sp.Y-125를 20ml씩 접종하여 28℃에서 3일간 배양한 다음, 원심분리하여 균체와 배양액을 분리하였다.2

Add 5 ml of shaking flasks, pH 7.0 medium consisting of 2% soluble starch, 0.5% polypeptone, 0.5% broth and 0.3% sodium chloride, and sterilize them at 121 ° C for 15 minutes, and then sterilize the bacteria solution Streptomyces sp. 20 ml of Y-125 was inoculated and incubated at 28 ° C. for 3 days, followed by centrifugation to separate the cells and the culture medium.

1550 ml of the 218AIU / ml culture solution thus obtained was concentrated and filtered to 1/10 under 60-70 ° C reduced pressure, and then precipitated impurities were removed and treated with 90% methanol to remove the precipitate, and then concentrated to obtain a concentrate.

This was dissolved in distilled water and freeze-dried for 36 hours to obtain 8.2 g amylase inhibitor of 5.5 × 10 ⁵ AIU / g.

Example 5

진탕 플라스크 5개에 1% 옥수수전분, 0.5% 펩톤, 0.5% 육즙, 0.3% 염화나트륨, pH 7.0인 배지를 각각 400ml씩 넣고 121℃, 15분간 살균한 후, 여기에다 미리 배양한 균액 스트렙토 마이세스 sp.Y-125 20ml를 각각 접종하여 28℃에서 4일간 배양하였다.2

400 ml of 1% corn starch, 0.5% peptone, 0.5% broth, 0.3% sodium chloride, pH 7.0 in 5 shake flasks were sterilized at 121 ° C. for 15 minutes, followed by precultured bacteria streptomyces sp. Each 20 ml of Y-125 was inoculated and incubated at 28 ° C. for 4 days.

Subsequently, the cells and the culture solution were centrifuged to obtain 1600 ml of a culture solution of 186AIU / ml. The culture solution was concentrated to 1/10, and the precipitate was filtered. Got water. This concentrate was dissolved in distilled water and lyophilized for 42 hours to obtain 9.8 g of amylase inhibitor, which was 5 × 10 ⁵ AIU / g.

Example 6

In Example 5, the culture medium was cultured under the same conditions using a medium containing 2% corn starch instead of 1% corn starch, and the culture solution was 220 AlU / ml, and the amylase inhibitor was 12.0 g (5.8 × 10 ⁵ AIU / g). Got it.

Example 7

진탕 플라스크 5개에 2.5% 가용성전분, 0.5% 폴리펩토, 0.5% 효모엑기스, 0.3% 염화나트륨, pH 6.0으로 조절한 배지를 각각 400ml씩 넣고, 이를 121℃에서 15분간 살균한 후, 미리 배양한 균액 스트렙토마이세스 sp.Y-125 20ml를 접종한 다음, 28℃에서 3일간 배양하였다.2

400 ml of medium adjusted with 2.5% soluble starch, 0.5% polypepto, 0.5% yeast extract, 0.3% sodium chloride, pH 6.0 was added to five shake flasks, and sterilized for 15 minutes at 121 ° C., followed by preculture 20 ml of Streptomyces sp. Y-125 was inoculated and then incubated at 28 ° C. for 3 days.

Subsequently, the culture was centrifuged to separate the cells and the culture solution, thereby obtaining 1550 ml of a culture solution of 2750 AIU / ml.

The culture solution was concentrated under reduced pressure at 60-70 ° C. to obtain 200 ml of a concentrated solution, which was filtered to remove impurity precipitates, and then treated with 90% ethanol to remove the precipitates, and the filtrate was concentrated and lyophilized for 24 hours. × 10 ⁵ of the inhibitor was obtained AIU / g. The inhibitor was again dissolved in distilled water and gel filtered using Sephadex G-10 column (1.8 × 30 cm).

At this time, elution was performed using distilled water, and only the active part was collected and lyophilized to obtain 0.8 g of an amylase inhibitor of 2.1 × 10 ₃ AIU / mg.

Example 8

진탕 플라스크 10개의 넣고, 살균후 배양하여 3000AIU/ml인 3000ml의 배양액을 얻었다.2 medium of the same conditions as in Example 7

Ten shake flasks were added, sterilized and cultured to obtain 3000 ml of the culture solution of 3000 AIU / ml.

Subsequently, the obtained culture solution was concentrated to 300 ml, treated with 90% ethanol to remove the precipitate, and the filtrate was concentrated to obtain a concentrate.

The concentrate was again dissolved in distilled water and lyophilized to obtain 15.5 g of an inhibitor of 7.1 × 10 ⁵ AIU / g, which was then dissolved in distilled water and gel filtered through a Sephadex G-10 column (2.1 × 30 cm). After lyophilization of only the active part, 1.02 g of an amylase inhibitor of 2700 AIU / mg was obtained.

Example 9

Under the same culture conditions as in Example 8, 0.1% of an antifoaming agent (silicon A, manufactured by Sigma Co., Ltd.) was added, and as a result, 16.4 g of an inhibitor of 7.3 × 10 ⁵ AIU / g was obtained, and Sephadex G-10 column (2.1 X 30 cm) was used to obtain 1.2 g of an inhibitor of 3100 AIU / mg.

Example 10

진탕 플라스크 10개에다, 2.5% 가용성 전분, 0.5% 폴리펩톤, 0.5% 효모추출물, 0.3% 염화나트륨, 0.05%소포제, pH 6.0으로 조절한 배지를 400ml씩 넣고, 이를 살균하여 미리 배양한 균액 20ml를 접종한 후, 28℃에서 3일간 배양하여 얻어진 배양물을 원심 분리하여 5250AIU/ml의 배양액 3000ml를 얻었다.2

10 shake flasks, 2.5% soluble starch, 0.5% polypeptone, 0.5% yeast extract, 0.3% sodium chloride, 0.05% antifoaming agent, 400 ml each of the medium adjusted to pH 6.0, sterilized and inoculated with 20 ml of the previously cultured bacteria solution After that, the culture obtained by culturing at 28 ° C. for 3 days was centrifuged to obtain 3000 ml of a culture solution of 5250AIU / ml.

The culture was concentrated to 1/10 at 60-70 ° C. under reduced pressure (10-50 mHg), filtered to remove impurities, which was then treated with 90% ethanol to remove precipitates, and then concentrated until complete removal of ethanol. .

The concentrate was then lyophilized to obtain an amylase inhibitor of 7.5 × 10 ⁵ AIU / g, which was gel filtered with a Sephadex G-10 column (1.8 × 40 cm) to give 1.70 g of an amylase inhibitor of 3300 AIU / mg.

Blood Glucose Change Characteristics

Animal experiments were conducted using the amylase inhibitors obtained in Example 10. The animals used were mice, and 5 mice were used as a group to induce hyperglycemia, and changes in blood glucose levels were observed at 5,15,30 minute intervals for these mice.

To induce hyperglycemia, 2.5 g of starch or maltose / kg mice were orally administered, and in the case of sugar, 5.0 g of sugar was also measured orally.

The blood glucose level was measured by the glucose-oxidase method.

A) Effect on starch degrading enzyme

B) Effect on maltose degrading enzyme

C) Effect on sugar degrading enzymes

Toxicity Test

Toxicity test was performed using the amylase inhibitor obtained in Example 10. The animals used were 17-20 g of mice and amylase inhibitors were observed using concentrations of 3,000-3,000,000 AIU / kg mice. There were no dead mice and no particular symptoms were found.

Weight change test

The amylase inhibitors obtained in Example 10 were mixed in the mouse feed by concentration to measure the change in the weight of the mice after administration.

The weight of the mice used was 17-20 g, and the weight change was observed for about 60 days with 5 dogs as a group. At 3,000 AIU / g (feed), weight loss was gradually observed after 42 days, and 9,000 AIU / g In 21 days, weight loss was observed.

Table 5 shows the weight change of the mouse.

TABLE 5

On the other hand, the amylase inhibitor obtained in Example 10 has the following characteristics.

1) Molecular Weight

In order to measure the molecular weight of the amylase inhibitor, the amylase inhibitor was gel filtered with Sephadex G-25 and developed by thin layer chromatography, and the molecular weight was 700-1500.

(Developing solvent: ethyl acetate: metalol: water = 27: 15: 23)

2) pH stability

The amylase inhibitor was added to the solution up to pH 2-12, reacted at 37 ° C. for 30 minutes, and the activity of the human salivary amylase was measured. As a result, all were stable in acidic, neutral and basic conditions (see FIG. 1). ).

3) thermal stability

1% amylase inhibitor was added to neutral solution at pH 7.0, heated at 100 ° C for 30-120 minutes, cooled, and measured for human activity on saliva amylase. Edo showed 85% activity (see Figure 2).

4) inhibitory effect on other amylase

As a result of investigating the inhibitory characteristics of amylose produced by microorganisms such as bacteria and fungi as well as human saliva amylase, it shows less than 50% inhibition against microbial amylase as shown in Table 6 below. Animal amylose, such as pancreas or amyloid of human saliva, had a significant inhibitory effect of more than 90%.

TABLE 6

± = 0-50% inhibition + = 90% abnormal

5) Chemical Properties of Amylase Inhibitors

The chemical properties of the inhibitors were positive for the Elson-Morgan reaction, as shown in Table 7 below, and the infrared absorption spectrum was similar to that of the polysaccharide (Oligosaccharid) absorption, and the melting point was also similar to maltopentose. See also 3)

In addition, it can be seen that the reducing sugar measurement after hydrolysis with hydrochloric acid is significantly increased.

As a result, the amylase inhibitor is a 3-6 sugar compound, and the pure sugar is an ether ether, which is considered to be a sugar and amino acid combined form.

It is soluble only in water and insoluble in methanol and ethanol.

TABLE 7

Claims (4)

Streptomyces sp. Y-125 (KCTC 8387P), characterized by producing amylase inhibitors. Amylase inhibitors isolated from cultures of Streptomyces sp.Y-125 (KCTC 8387P) strain. A method for producing an amylase inhibitor, comprising culturing Streptomyces sp.Y-125 (KCTC 8387P) strain and separating the amylase inhibitor from the culture. The amylase inhibitor is characterized in that the molecular weight of 700 to 1500, it is stable at pH 2 to 12.

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