KR20000025032A - Preparation method of acarviosine-glucose - Google Patents

Abstract

PURPOSE: A preparation method of high-purity acarviosine-glucose is provided which enables the title compound to be produced in commercial quantity without a byproduct and a complicated purification process. CONSTITUTION: A process for the production of acarviosine-glucose as a derivative of acabose comprises hydrolyzing acarbose by acarbose-degradable amylase and optionally yeast-fermenting simultaneously with acarbose hydrolysis or after acarbose hydrolysis. In this process, the acarbose-degradable amylase is amylolytic enzymes which principally produce maltose by hydrolyzing starch, principally produce pannose by decomposing pullulan and principally produce maltose by decomposing cyclodextrin. The title compound capable of effectively inhibiting carbohydrate can be produced in commercial quantity by the process without a byproduct and complicated purification process.

Description

Method for preparing acabiocin-glucose

The present invention relates to a method for producing pseudosamtanose which inhibits carbohydrate degrading enzyme.

Acarbose, a representative compound that inhibits carbohydrate degrading enzymes, is a pseudotetrasaccharide composed of unsaturated cyclitol molecules, amino sugars, and two molecules of glucose, which are no longer caused by carbohydrate degrading enzymes. It is reported that it is not degraded, and is used as an inhibitor of carbohydrate degrading enzymes such as α-glucosidase, glucoamylase, α-amylase and cyclodextrin glucotransferase (hereinafter CGTase). Therefore, it effectively inhibits α-glucosidase, an enzyme secreted by the small intestine, and has an effect of inhibiting glucose intake.

Acabos

In the structure of acarbose and its derivatives, unsaturated cyclitol and amino sugars are known to be essential for carbohydrate degrading inhibitory activity, and their specificity is determined by the number of bound glucose. In general, it is known that acarbose, in which two glucoses are bound, has the greatest inhibitory effect on α-glucosidase, and the greater the number of bound glucose, the greater the inhibitory effect on α-amylase. In in vivo experiments, acarviosine-glucose with only one molecule of glucose was reported to have the highest inhibitory effect on sucrose and amylase (DD Schmidt, W. Frommer, B. Junge, L. M ller, W. Wingender and E. Truscheit (1980) in First International Symposium on Acarbose (Creutzfeldt, W., Ed.) pp 5-15, Excerpta Medica, Amsterdam).

Acarbiosine-glucose

Acabiocin-glucose is a pseudosamtanose obtained by hydrolysis of a molecule of glucose at the reducing end of acarbose, and has an inhibitory effect on carbohydrate degrading enzymes such as acarbose. Acabiocin-glucose has been reported to be produced by heating acarbose with dilute sulfuric acid at 100 ° C. for 1 hour to remove glucose molecules (B. Junge, H. B.). shagen, J. Stoltefuβ, L. M ller (1980) in Enzyme inhibitors (Brodbeck, U., Ed.) pp. 123-137, Verlag Chemie, Weinheim). However, this method is a chemical decomposition method that is not highly specific and may cause various side reactions and requires a complicated process of purifying acabiocin-glucose after the reaction.

It is an object of the present invention to provide a method for producing acarbiocin-glucose that inhibits carbohydrate degrading enzymes.

It is still another object of the present invention to provide a method for preparing acarbiosin-glucose that inhibits carbohydrate degrading enzymes with high purity.

1 is a thin layer chromatography photograph for the analysis of acabiocin-glucose.

The present invention relates to a method for preparing acabiocin-glucose.

The method of the present invention consists in hydrolyzing acarbose to acarbose degradable amylase.

The present inventors hydrolyze starch to produce mainly maltose and a small amount of glucose, and to break down pullulan to produce pannose, and to decompose cyclodextrin to produce mainly maltose and a small amount of glucose. It has been found that carbose hydrolyzes into glucose and acabiocin-glucose.

In the present specification, the acarbose degradable amylase hydrolyzes starch to produce mainly maltose, and decomposes pullulan to produce phanose, mainly to decompose cyclodextrin to produce maltose, and to convert acarbose to glucose It refers to an enzyme that breaks down acabiocin-glucose.

For example, maltogenic amylase (hereinafter referred to as BSMA) produced from Bacillus stearothermophilus (KFCC-10896), reported in Korean Patent Application No. 96-25135, refers to glucose and acabiocin-glucose. Decompose into As another example, maltogenic amylases isolated from Bacillus licheniformis (ATCC 27811) reported in US Pat. These amylases are starch hydrolases that hydrolyze starch to produce maltose, break down pullulan to produce pannose, and decompose cyclodextrin to produce maltose.

The method of the present invention consists in hydrolyzing acarbose to acarbose degradable amylase.

In the method according to the present invention, the acarbose is dissolved in a suitable buffer, and then reacted at an optimum temperature of the acarbose degradable amylase for the acarbose hydrolysis.

Another method of the present invention relates to a method for preparing high-purity acabiocin-glucose consisting of yeast fermentation simultaneously with or after acavo hydrolysis by acavo degradable amylase. .

Thus, by the yeast fermentation at the same time or after the acarbose hydrolysis by the acarbose degradable amylase, the glucose produced by the akabose hydrolysis can be removed so that high purity aka Biosine-glucose can be prepared.

The kind of yeast used by the method of this invention is not specifically limited, Any yeast which can use glucose as a carbon source can be used. Yeast fermentation can be carried out according to conditions well known in the yeast fermentation field, and will depend on the type of yeast used.

Hereinafter, the present invention will be described through examples, and the scope of the present invention is not limited by these examples.

Example 1 Preparation of Acarbiocin-Glucose

Amylose-producing strains deposited with LB medium were inoculated in LB medium, pre-incubated at 37 ° C, and incubated in the same medium for 10 hours. Isolation gave cells. The recovered cells were suspended in 50 mM Tris-Cl buffer solution of ph 7.5 and then disrupted by ultrasonication and centrifuged to obtain supernatant. The supernatant was partitioned with ammonium sulfate and purified by column chromatography through DEAE-TOYOPEARL and Mono Q columns to obtain purified amylase.

A 10% (w / v) solution in which acabose was dissolved in a 50 mM citrate buffer solution (ph 6.0) was left in a 40 ° C. water bath for 10 minutes and then added with 160 Cu BSMA per mg of acabose for 17 hours. The first reaction was carried out. For complete hydrolysis of acarbose, BSMA was added to the enzyme reaction solution under the same conditions as the first reaction, followed by a second reaction for 17 hours. After the second reaction, the reaction solution was heated in boiling water for 5 minutes to terminate the reaction.

Example 2 Preparation of Acarbiocin-Glucose

ThMA amylase-producing strains were inoculated in LB medium, pre-incubated at 37 ° C, incubated for 10 hours in the same medium, and centrifuged to obtain cells. The recovered cells were suspended in 50 mM Tris-Cl buffer at pH 7.5 and then destroyed by ultrasonication and centrifuged to obtain supernatant. The supernatant was fractionated with ammonium sulfate and purified by column chromatography through Q-Sepharose and DEAE 8HR columns to obtain purified amylase.

A 10% (w / v) solution in which acarbose was dissolved in 50 mM acetate buffer (ph 6.0) was previously left in a 40 ° C. water bath for 10 minutes, and then 160 Cu of ThMA per 1 mg of akaboose was added for 1 hour. Tea reaction. For complete hydrolysis of acarbose, ThMA was added to the enzyme reaction solution under the same conditions as the first reaction, followed by secondary reaction for 17 hours. After the second reaction, the reaction solution was heated in boiling water for 5 minutes to terminate the reaction.

Example 3 Preparation of High Purity Acabiocin-Glucose

First, in order to activate the yeast used for the reaction, baking in granular form in YPD medium (yeast extract 1% (w / v), bactopeptone 2% (w / v), dextrose 2% (w / v)) Yeast was inoculated and incubated at 27 ° C. for 18 hours with shaking incubator.

Instead of using 160 Cu BSMA per 1 mg of acarbose in Example 1 to produce high purity acarbiocin-glucose, the same as Example 1 using 160 Cu BSMA per 1 mg of acarbose and 2 μL of yeast culture It was reacted by the method. After the second reaction, the reaction solution was heated in boiling water for 5 minutes to terminate the reaction.

Example 4 Analysis of Reaction Products Using Thin Film Chromatography

After completion of the reaction, the reaction product was analyzed using thin layer chromatography and is shown in FIG. 1. A is a maltooligosaccharide standard (G1-G7), B is the acarbose hydrolyzate obtained in Example 1 (from above, glucose, acabiocin-glucose, acarbose, isocarbose), C is an example The yeast culture obtained in Fig. 2 and the acarbose hydrolyzate containing both BSMA and D are the acarbose standards. Each sample was loaded 1 μL into a Whatman K5F TLC plate (20 cm × 20 cm) and developed twice in a developing solution with a 3: 1: 1 (v / v / v) ratio of isopropyl alcohol, ethyl acetate and distilled water. After development, the plate was well dried, immersed in a coloring solution in which 0.3% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (v / v) sulfuric acid was dissolved in methanol, and then taken out in a 110 ° C oven. 10 minutes of color development. BSMA hydrolyzed acarbose to produce acarbiocin-glucose and glucose, and a significant amount of glucose and isocarbose remained in the reaction without yeast (FIG. 1B). However, when the yeast culture solution was added, glucose was completely removed and the amount of other by-products was small (Fig. 1C).

Example 5 Enzyme Inhibitory Characteristics of Acabiocin-Glucose

Ki values for each enzyme were determined to investigate the inhibitory properties of acarbiocin-glucose against carbohydrate hydrolase.

The enzymes used were α-glucosidase from baker's yeast, α-glucosidase from rat, α-amylase from pig pancreas, and Bacillus marcrans CGTase. .

Α-glucosidase

Maltose was used as the substrate and glucose hydrolyzed by the enzyme was quantified on a spectrophotometer by glucose oxidase-peroxidase enzyme method. That is, 0.6 mL of substrate, 50 μL of color reagent (4-aminoantipyrine), 50 μL of color enzyme (glutose oxidase, peroxidase), 50 μL of enzyme inhibitor (acarbose or acabiocin-glucose) in cuvette 50 μL of glucosidase was added and absorbance was continuously measured at 500 nm, and the reaction temperature was maintained at 37 ° C. The α-glucosidase added at this time was left in the enzyme inhibitor solution for 5 minutes before the reaction. The reaction rate was determined by the average slope of absorbance change for 10 minutes.

2. α-amylase

Soluble starch was used as a substrate and reacted in a 37 ° C. constant temperature water bath using a 50 nM phosphate buffer solution having a pH of 7.0 to which 6 mM NaCl was added. Substrate concentrations were used at 0.0444, 0.0889, 0.444, 1.07 mg / ml, respectively. The reaction was performed by adding 100 μL of enzyme inhibitor (acarbose, acabiocin-glucose) and 50 μL of α-amylase to a 1200 μL substrate. 100 μl of the reaction solution was taken for each hour, and the reaction was stopped by placing in a microplate reader containing a color reagent. After the color development at 85 ° C. for 35 minutes, the absorbance was measured at 540 nm to determine the initial reaction rate.

As a color development method, a copper-bicinchoninate reducing value method was used. 97.1 g of disodium 2,2'-bicinconate, 3.2 g of sodium carbohydrate monohydrate, 1.2 g of sodium bicarbonate was dissolved in distilled water to 50 ml, 62 mg of divalent copper sulfate, 63 mg of L-serine was dissolved in distilled water, and the solution B adjusted to 50 ml was used by mixing 1: 1.

3. CGTase

Maltodextrin (average Dp = 13) was used as a substrate and pH 6.0 50 mM imidazole solution to which 1 mM calcium chloride was added was used as a buffer for enzymatic reaction in a 25 ° C. constant temperature water bath. An initial reaction rate was determined by continuously measuring absorbance reduction at 528 nm while adding 100 μL of enzyme solution to a mixture of 400 μL 0.5 mM methyl orange solution and 500 μL substrate solution. The amount of cyclodextrin produced was determined from a quantitative curve prepared by measuring the degree of decrease in absorbance when α-cyclodextrin forms a clathrate compound with methyl orange.

Ki values for each enzyme are shown in Table 1. Acabiocin-glucose showed a significantly lower Ki value for α-glucosidase and a 6-fold lower Ki value for CGTase than acarbose. It was found to act as an inhibitor. The inhibitory effect on porcine pancreatic α-amylase was almost similar, and α-glucosidase isolated from the small intestine of the rat showed a smaller Ki value than acarbose.

According to the present invention, it is possible to produce a large amount of acabiocin-glucose that can effectively inhibit carbohydrate degrading enzymes.

Claims (3)

A method for producing acarbiosine-glucose, which comprises hydrolyzing acarbose to acarbose degradable amylase. The method of claim 1, wherein the acarbose is hydrolyzed simultaneously with or hydrolyzed to the acarbose degradable amylase and further removed yeast fermentation to remove glucose produced by the acarbose hydrolysis. The method according to claim 1 or 2, wherein the acarbose degradable amylase is an amylase produced from Bacillus stearothermophilus KFCC-10896.