JPH06277087A - Production of glycoside - Google Patents

Abstract

PURPOSE:To obtain a glycoside of a phenolic OH group-containing compound by treating a phenolic OH group-containing compound and a specific glucan with a bacterium-derived saccharification type alpha-amylase. CONSTITUTION:(A) A phenolic OH group-containing compound (hydroquinone or dimethoxyphenol) and (B) a glucan containing an alpha-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various kinds of starches are treated with (C) a bacterium-derived saccharification type alpha-amylase [e.g. saccharification type alpha-amylase derived from Bacillus subtilis X-23 (FERM P-13,560) and saccharification type alpha-amylase derived from IFO14,140]. A glycoside of a flavonoid analogous compound is obtained when the flavonoid analogous compound is used as a receptor instead of the component A.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing glycosides.

[0002]

2. Description of the Related Art Enzymes of a compound having a phenolic OH group or a flavonoid analogue and a glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches. There are no detailed studies on the production of glycosides by the action of. For example, Amylase Symposium Vol, 101
975, pp. 81-89, a study of transferring another saccharide to another saccharide to produce another saccharide is reported. However, there are no studies on enzymes that perform glycosyl transfer to phenol OH groups in compounds and OH groups in flavonoid analogs. Further, it is not known that a compound having a phenolic OH group or a flavonoid analog compound becomes a glycoside by the action of a saccharified amylase derived from bacteria.

[0003]

The compound having a phenolic OH group used in the present invention is hydroquinone, dimethoxyphenol or the like. The flavonoid analogs used in the present invention include flavones, isoflavones, flavonols,
Examples thereof include flavanone, flavanonol, catechin, aurone, anthocyanidin, chalcone and dihydrochalcone. Bacterial-derived glycated α-amylase (hereinafter referred to as the enzyme group of the present invention) includes, for example, Bacillus subtilis X-23 (Deposit No. P-13560, Institute of Industrial Science and Technology) and IFO14140.
There are glycated α-amylase (hereinafter referred to as amylase BSA) and the like. The method for producing the glycoside obtained by the present invention is as follows. Compounds having a phenolic OH group such as hydroquinone or dimethoxyphenol, or flavones, isoflavones, flavonols, flavanones,
Flavanon analogues such as flavanonol, catechin, aurone, anthocyanidin, chalcone or dihydrochalcone 1 to 20%, preferably 1 to 5%, and glucan 1 having α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches 1 The enzyme group of the present invention is added to a solution containing -20%, preferably 5-10%, and allowed to act at normal temperature and time to obtain a reaction solution. A purified preparation of glycoside can be obtained from this reaction solution by partitioning with various solvents and purification by various chromatography.

Amylase X-23 has the following physicochemical properties. 1. Action If no receptor such as a phenol analog or a flavonoid analog is present, it decomposes (hydrolyzes) glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin, and various starches. However, if a glucan having an α-1,4 bond such as a phenol analog or a flavonoid analog and a maltooligosaccharide, amylose, amylopectin, and various starches is present at the same time, α-1 such as maltooligosaccharide, amylose, amylopectin, and various starches can be used. Glucan having 4 bonds is decomposed, and glycosyl transfer is performed to the OH group in the phenol analog or flavonoid analog by α bond.

2. Receptor specificity Phenolic OH group or flavone in phenol related compounds such as hydroquinone or dimethoxyphenol,
Glycosyl transfer is carried out by α-bonding to the OH group in the flavonoid analog compound such as isoflavone, flavonol, flavanone, flavanonol, catechin, aurone, anthocyanidin, chalcone or dihydrochalcone.

3. Optimum pH and stable pH When hydrolyzing soluble starch, the optimum pH is pH 6
~ 7 and stable pH is pH 5-8 (Figure 1). When transferring sugar to hydroquinone, the optimum pH is pH 5.
~ 8 and stable pH is pH 4-13 (Figure 2).

4. Titer measurement method (α-amylase activity measurement method) Amylase activity is measured as follows. To 50 μl of the enzyme solution incubated at 40 ° C., 200 μl of 1.5% soluble starch (adjusted to pH 5.5 with 40 mM acetic acid-Na buffer solution) incubated at 40 ° C. was added, and 4
Incubate for 10 minutes at 0 ° C. The reaction is stopped by adding 2 ml of a 5: 1 mixture of 0.5N acetic acid and 0.5N hydrochloric acid to this reaction solution and stirring. This solution 0.
1 ml was taken, 5 ml of a solution containing 0.005% iodine and 0.05% potassium iodide was added and stirred,
Leave at room temperature for 20 minutes. The absorbance at 660 nm of this solution is measured, and this absorbance is designated as C. Separately, as a blank, a solution using purified water instead of the above enzyme solution was prepared, and the absorbance obtained by performing the same operation was designated as B. From C and B thus obtained, the amylase activity A is obtained by the following formula. A = (B−C) / B × 10 However, 1 unit of amylase activity is assumed to be 10% of B value.

(Measuring Method of Glycosyl Transfer Rate) The glycosyl transfer rate is measured as follows. A solution containing 10% of a donor such as soluble starch or maltopentaose and 2% of an acceptor of a compound having a phenolic OH group such as hydroquinone or catechin and an enzyme solution were mixed at a ratio of 1: 1, and the mixture was mixed at 40 ° C. for 1 hour.
Allow to react for 6-24 hours. The reaction solution was diluted 15 times and HP
The analysis is performed by LC (using an ODS column RP-18 manufactured by MERCK, using an eluent of 10% methanol adjusted to pH 2.5). Detection is by absorbance at 280 nm. As a control, a solution using purified water instead of the enzyme solution is prepared and the same operation as above is performed. The glycosyl transfer rate is calculated by the following formula. Glycosyl transfer rate = peak area of glycoside / peak area of control receptor x 100

5. Suitable temperature range for action Stable glycosyl transfer from 30 ° C to 70 ° C at pH 5.5 (Fig. 3).

6. Inhibitors Inhibited by copper and zinc ions, but hardly inhibited by EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion (FIG. 4).

7. Conditions of deactivation: Complete deactivation when treated at 100 ° C for 15 minutes.

8. Purification method After removing the bacterial cells from the culture solution, salting out with ammonium sulfate (80% saturation) is performed and the resulting precipitate is dissolved and dialyzed. This was subjected to Q-Sepharose column chromatography (pH
Dialysis is carried out). Phenyl Sepharose column chromatography (using 0.8M-0M ammonium sulfate concentration gradient)
After dialysis, dialysis is performed. The dialyzed solution is subjected to gel filtration with Superose 12 and freeze-dried to obtain a purified sample. The present enzyme purified by such a method shows a single band on SDS-polyacrylamide gel electrophoresis.

9. It has a molecular weight of about 65,000 (by SDS-polyacrylamide gel electrophoresis).

10. Ultraviolet absorption spectrum The maximum absorption is 267 nm and the minimum absorption is 252 nm (FIG. 5).

11.7 Minoic Acid Analysis The amino acid composition is shown in FIG.

Amylase BSA has the following physicochemical properties. 1. Action If no receptor such as a phenol analog or a flavonoid analog is present, it decomposes (hydrolyzes) glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin, and various starches. However, if a glucan having an α-1,4 bond such as a phenol analog or a flavonoid analog and a maltooligosaccharide, amylose, amylopectin, and various starches is present at the same time, α-1 such as maltooligosaccharide, amylose, amylopectin, and various starches can be used. Glucan having 4 bonds is decomposed, and glycosyl transfer is performed to the OH group in the phenol analog or flavonoid analog by α bond.

2. Receptor specificity The phenolic OH group in a phenol analog compound such as hydroquinone or dimethoxyphenol, or the OH group in a flavonoid analog compound such as flavone, isoflavone, flavonol, flavanone, flavanonol, catechin, aurone, anthocyanidin, chalcone or dihydrochalcone. , Glycosylation is performed by α bond.

3. Optimum pH and stable pH When soluble starch is hydrolyzed, the optimum pH is pH.
It is 5.3, and the stable pH is pH 4.5 to 9.2. When sugar transfer to hydroquinone, the optimum pH is p
H6 and stable pH is pH 5-8.

4. Titer measuring method The same as the titer measuring method for amylase X-23.

5. Suitable temperature range for action Stable glycosyl transfer at 30 to 60 ° C at pH 5.5.

6. Inhibitor Although it is inhibited by copper ion and zinc ion, it is hardly inhibited by EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion.

7. Conditions of deactivation: Complete deactivation when treated at 100 ° C for 15 minutes.

9. It has a molecular weight of about 47,000.

[0024]

[Action] The action of the enzyme group of the present invention is as follows. 1. Decomposition of Starch The enzyme group of the present invention hydrolyzes starch to produce glucose, maltose, maltotriose and branched oligosaccharides. 2. Sugar transfer The enzyme group of the present application decomposes α-1,4 glucans such as maltooligosaccharides to produce glucose, maltose, and maltotriose, and also converts glucose, maltose to OH groups in phenolic OH groups and flavonoid analogs. ,
Transfers sugars such as maltotriose with α bond.

[0025]

【Example】

(Example 1) 10% hydroquinone and 20% maltopentaose were dissolved in a 20 mM acetate buffer (adjusted to pH 5.5). Amylase activity 6 was added to 10 ml of this solution.
10 ml of enzyme solution of amylase X-23 having 0 units was added. After reacting the above solution at 40 ° C. for 16 hours, 3-fold amount of ethyl acetate was added and shaken vigorously. After standing at room temperature for 30 minutes, the aqueous phase fraction was collected.
This operation was repeated 3 times to completely remove unreacted hydroquinone, and then dried under reduced pressure. The glycoside was adsorbed on an activated carbon column in which the sample was dissolved in purified water and equilibrated with purified water. Next, the unreacted sugar adsorbed on the activated carbon column was eluted with 20% methanol, and then the glycoside was eluted with 100% methanol. The 100% methanol fraction was dried under reduced pressure, then dissolved again in purified water and freeze-dried. About 200 mg of hydroquinone glycoside was obtained by the above procedure.
I was able to get it. The purified sample was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the HPLC analysis shown in the above method for measuring the glycosylation rate, to find the peak of hydroquinone glycoside (hereinafter referred to as glucosylhydroquinone). Disappeared completely and a new hydroquinone peak appeared. As a result, glucosyl hydroquinone is converted into hydroquinone with glucose α
It was found to be a linked glycoside. Furthermore, the structure of glucosyl hydroquinone was analyzed by NMR (JNM-GX270,
When confirmed by JEOL), the same result as above was confirmed (FIG. 8). The result of NMR analysis is shown in FIG.
It was shown to. The glycosyl transfer rate due to this action was about 27%. Further, after 6 to 15 hours from the start of the reaction, it was possible to further increase the transglycosylation rate by adding a new donor such as maltopentaose. When 6 hours after the start of the reaction, maltopentaose was added to 5%, the transglycosylation rate was about 40%.

Example 2 Hydroquinone 10% and maltopentaose 20% were added to 20 mM acetate buffer (pH).
(Prepared to 5.5). To 10 ml of this solution,
Amylase BSA10 having 60 units of amylase activity
ml was added. After the above solution was reacted at 40 ° C. for 16 hours, 3-fold amount of ethyl acetate was added to the reaction solution and shaken vigorously. After standing at room temperature for 30 minutes, the aqueous phase fraction was collected. This operation was repeated 3 times to completely remove unreacted hydroquinone, and then dried under reduced pressure. The glycoside was adsorbed on an activated carbon column in which the sample was dissolved in purified water and equilibrated with purified water. After elution of unreacted sugar adsorbed on the activated carbon column with 20% methanol, 100
The glycoside was eluted with% methanol. The 100% methanol fraction was dried under reduced pressure, then dissolved again in purified water and freeze-dried. By the above operation, about 150 mg of hydroquinone glycoside could be obtained. The purified sample was treated with α-glucosidase (manufactured by TOYOBO), and the HPLC was shown in the method for measuring the glycosidation rate.
As a result of the analysis, the peak of glucosylhydroquinone disappeared completely and the peak of hydroquinone newly appeared. From this, it was found that glucosylhydroquinone is a glycoside in which glucose is α-bonded to hydroquinone. The glycosyl transfer rate due to this action was about 22.
%Met. Further, the transglycosylation rate could be further increased by adding maltopentaose as a donor to the above reaction solution 6 to 15 hours after the start of the reaction. 6 hours after the start of the reaction, the transglycosylation rate was about 32% when maltopentaose was added to 5%.

Example 3 Catechin 2% and maltopentaose 10% were dissolved in a 20 mM acetate buffer (adjusted to pH 5.5). To 1 ml of this solution, 1 ml of enzyme solution of amylase X-23 having 6 units of amylase activity
Was added and the reaction was carried out at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (ODS column RP-18
Was eluted with a solution consisting of 12 parts, 2 parts, and 86 parts of acetonitrile and ethyl acetate 0.05% phosphoric acid.
The peak of the glycoside of catechin was newly confirmed in addition to the unreacted catechin. Furthermore, regarding this reaction solution, α-glucosidase (TOY
When the same HPLC analysis was carried out after treatment with OBO), the peak of the glycoside of catechin disappeared and the peak of unreacted catechin increased. From this, it was confirmed that the glycoside of catechin was a compound in which glucose was α-bonded to catechin. The glycosyl transfer rate in this reaction was about 25%.

Example 4 2% catechin and 10% maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). To 1 ml of this solution, 1 ml of an enzyme solution of amylase BSA having 6 units of amylase activity was added and reacted at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (using an ODS column RP-18, eluted with a solution consisting of 12 parts, 2 parts and 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid).
The peak of the glycoside of catechin was newly confirmed in addition to the unreacted catechin. Furthermore, regarding this reaction solution, α-glucosidase (TOY
When the same HPLC analysis was carried out after treatment with OBO), the peak of the glycoside of catechin disappeared and the peak of unreacted catechin increased. From this, it was confirmed that the glycoside of catechin was a compound in which glucose was α-bonded to catechin. The glycosyl transfer rate in this reaction was about 22%.

Example 5 2% epigallocatechin and 10% maltopentaose were added to a 20 mM acetate buffer (pH).
(Prepared to 5.5). To 1 ml of this solution,
1 ml of an enzyme solution of amylase X-23 having a unit of amylase activity was added, and the reaction was carried out at 40 ° C for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, acetonitrile, ethyl acetate, and 0.1%).
It was eluted with a solution consisting of 12 parts, 2 parts and 86 parts and was detected at 280 nm with 05% phosphoric acid). As a result, a new glycoside peak of epigallocatechin was confirmed in addition to unreacted epigallocatechin. Furthermore, when this reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of epigallocatechin disappeared and the peak of unreacted epigallocatechin increased. did. From this, it was confirmed that the glycoside of epigallocatechin was a compound in which glucose was α-bonded to epigallocatechin. The glycosyl transfer rate in this reaction was about 28%.

Example 6 Epigallocatechin 2% and maltopentaose 10% were added to 20 mM acetate buffer (pH).
(Prepared to 5.5). To 1 ml of this solution,
1 ml of an enzyme solution of amylase BSA having a unit of amylase activity was added, and the reaction was carried out at 40 ° C for 16 hours. The reaction solution is analyzed by HPLC (ODS column R
Using P-18, acetonitrile, ethyl acetate and 0.0
5% phosphoric acid was eluted with a solution consisting of 12 parts, 2 parts and 86 parts and was detected at 280 nm), and a new glycoside peak of epigallocatechin was confirmed in addition to unreacted epigallocatechin. Furthermore, regarding this reaction solution,
When the same HPLC analysis was carried out after treatment with α-glucosidase (manufactured by TOYOBO), the peak of the glycoside of epigallocatechin disappeared and the peak of unreacted epigallocatechin increased. From this, it was confirmed that the glycoside of epigallocatechin was a compound in which glucose was α-bonded to epigallocatechin. The glycosyl transfer rate in this reaction was about 25%.

Example 7 2% of epicatechin gallate and 10% of maltopentaose were added to 20 mM acetate buffer (p
(Prepared to H5.5). To 1 ml of this solution,
1 ml of an enzyme solution of amylase X-23 having 6 units of amylase activity was added and reacted at 40 ° C for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, eluted with a solution consisting of 12 parts, 2 parts and 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid, and detected by 280 nm). In addition to epicatechin gallate in the reaction, a new peak of epicatechin gallate glycoside was confirmed. Furthermore, when this reaction liquid was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of epicatechin gallate disappeared and the peak of unreacted epicatechin gallate increased. did.
From this, it was confirmed that the epicatechin gallate glycoside was a compound in which glucose was α-bonded to epicatechin gallate. The transglycosylation rate in this reaction is about 1
It was 3%.

Example 8 2% of epicatechin gallate and 10% of maltopentaose were added to 20 mM acetate buffer (p
(Prepared to H5.5). To 1 ml of this solution,
1 ml of an enzyme solution of amylase BSA having 6 units of amylase activity was added and reacted at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, acetonitrile, ethyl acetate, and 0.1%).
It was eluted with a solution consisting of 12 parts, 2 parts, and 86 parts and was detected by 280 nm with 05% phosphoric acid). As a result, a new glycoside peak of epicatechin gallate was confirmed in addition to unreacted epicatechin gallate. Furthermore, when this reaction liquid was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of epicatechin gallate disappeared and the peak of unreacted epicatechin gallate increased. did. From this, it was confirmed that the epicatechin gallate glycoside was a compound in which glucose was α-bonded to epicatechin gallate. The transglycosylation rate in this reaction is about 10%.
Met.

(Example 9) Epigallocatechin gallate 2
% And 10% maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). 1 ml of this solution
Amylase X-2 having 6 units of amylase activity
3 ml of the enzyme solution was added and the reaction was carried out at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, eluted with a solution consisting of 12 parts, 2 parts and 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid, and detected by 280 nm). In addition to the reaction epigallocatechin gallate, a new peak of epigallocatechin gallate glycoside was confirmed. Furthermore, regarding this reaction solution, α-glucosidase (TOY
When the same HPLC analysis was carried out by treatment with OBO), the peak of the glycoside of epigallocatechin gallate disappeared and the peak of unreacted epigallocatechin gallate increased. From this, it was confirmed that the glycoside of epigallocatechin gallate was a compound in which glucose was α-bonded to epigallocatechin gallate. The glycosyl transfer rate in this reaction was about 12%.

Example 10 2% of epigallocatechin gallate and 10% of maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). 1m of this solution
Amylase BS having 6 units of amylase activity in l
1 ml of the enzyme solution A was added and reacted at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, eluted with a solution consisting of 12 parts, 2 parts and 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid, and detected by 280 nm). In addition to the reaction epigallocatechin gallate, a new peak of epigallocatechin gallate glycoside was confirmed. Furthermore, regarding this reaction solution, α-glucosidase (TOY
When the same HPLC analysis was carried out by treatment with OBO), the peak of the glycoside of epigallocatechin gallate disappeared and the peak of unreacted epigallocatechin gallate increased. From this, it was confirmed that the glycoside of epigallocatechin gallate was a compound in which glucose was α-bonded to epigallocatechin gallate. The glycosyl transfer rate in this reaction was about 8%.

Example 11 2% epicatechin and 10% maltopentaose were added to 20 mM acetate buffer (pH).
(Prepared to 5.5). To 1 ml of this solution,
1 ml of an enzyme solution of amylase X-23 having a unit of amylase activity was added, and the reaction was carried out at 40 ° C for 16 hours. This reaction solution was analyzed by HPLC (using ODS column RP-18, acetonitrile, ethyl acetate, and 0.1%).
It was eluted with a solution consisting of 12 parts, 2 parts and 86 parts and was detected at 280 nm with 05% phosphoric acid). As a result, a peak of a glycoside of epicatechin was confirmed in addition to unreacted epicatechin. Furthermore, when this reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of epicatechin disappeared and the peak of unreacted epicatechin increased. From this, it was confirmed that the glycoside of epicatechin was a compound in which glucose was α-bonded to epicatechin. The transglycosylation rate in this reaction is about 23%.
Met.

Example 12 2% epicatechin and 10% maltopentaose were added to a 20 mM acetate buffer (pH 5.
5)). 1 ml of this solution contains 1 unit of amylase BSA having 6 units of amylase activity.
ml was added and the reaction was carried out at 40 ° C. for 16 hours.
The reaction solution was analyzed by HPLC (ODS column RP-
18, using acetonitrile and ethyl acetate 0.05%
Phosphoric acid was eluted with a solution consisting of 12 parts by 2 parts and 86 parts by detection at 280 nm), and a new glycoside peak of epicatechin was confirmed in addition to unreacted epicatechin. Furthermore, when this reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of epicatechin disappeared and the peak of unreacted epicatechin increased. From this, it was confirmed that the glycoside of epicatechin was a compound in which glucose was α-bonded to epicatechin. The glycosyl transfer rate in this reaction was about 23%.

Example 13 3.4% dimethoxyphenol and 10% maltopentaose were dissolved in a 20 mM acetate buffer (adjusted to pH 5.5). This solution 1
Amylase x having 6 units of amylase activity per ml
-23 enzyme solution (1 ml) was added and the reaction was carried out at 40 ° C. for 16 hours. This reaction solution is analyzed by HPLC (OD
S column RP-18 was used to elute with 25% methanol and detected by 280 nm), and unreacted 3.
In addition to 4-dimethoxyphenol, a new peak of the glycoside of 3.4-dimethoxyphenol was confirmed. Furthermore, this reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. When the peak of the glycoside of 3.4 dimethoxyphenol disappeared and unreacted 3.4 dimethoxyphenol was detected. Peak increased. From this, it was confirmed that the glycoside of 3.4 dimethoxyphenol was a compound in which glucose was α-bonded to 3.4 dimethoxyphenol.

(Example 14) 3.5% dimethoxyphenol and 10% maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). This solution 1
Amylase X having 6 units of amylase activity in ml
1 ml of the enzyme solution of -23 was added and the reaction was carried out at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (O
By using a DS column RP-18 and eluting with 25% methanol and detecting at 280 nm), a peak of a glycoside of 3.5 dimethoxyphenol was confirmed in addition to the unreacted 3.5 dimethoxyphenol. further,
About this reaction solution, α-glucosidase (TOYOB
The same HPLC analysis was carried out and the peak of the glycoside of 3.5 dimethoxyphenol disappeared and the peak of unreacted 3.5 dimethoxyphenol increased. From this, it was confirmed that the glycoside of 3.5 dimethoxyphenol was a compound in which glucose was α-bonded to 3.5 dimethoxyphenol.

Example 15 2.3% dimethoxyphenol and 10% maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). This solution 1
Amylase X having 6 units of amylase activity in ml
1 ml of the enzyme solution of -23 was added and the reaction was carried out at 40 ° C. for 16 hours. This reaction solution was analyzed by HPLC (O
Using a DS column RP-18, eluting with 25% methanol and detecting at 280 nm), a new peak of the glycoside of 2.3 dimethoxyphenol was confirmed in addition to the unreacted 2.3 dimethoxyphenol. further,
About this reaction solution, α-glucosidase (TOYOB
When the same HPLC analysis was carried out after the treatment with a product manufactured by Company O), the peak of the glycoside of 2.3 dimethoxyphenol disappeared and the peak of unreacted 2.3 dimethoxyphenol increased. From this, it was confirmed that the glycoside of 2.3 dimethoxyphenol was a compound in which glucose was α-bonded to 2.3 dimethoxyphenol.

Example 16 Acetaminophenone 2%
And 10% maltopentaose were dissolved in 20 mM acetate buffer (adjusted to pH 5.5). 1 ml of this solution
Amylase X-2 having 6 units of amylase activity
1 ml of the enzyme solution of 3 was added and the reaction was carried out at 40 ° C. for 16 hours. This reaction solution is analyzed by HPLC (ODS
Elute with 25% methanol using column RP-18 2
(Detected by 80 nm), a new peak of acetaminophenone glycoside was confirmed in addition to unreacted acetaminophenone. Furthermore, when this reaction liquid was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis, the peak of the glycoside of acetaminophenone disappeared and the peak of unreacted acetaminophenone increased. did. From this, it was confirmed that the acetaminophenone glycoside was a compound in which glucose was α-bonded to acetaminophenone.

[0041]

EFFECT OF THE INVENTION By the enzyme group of the present invention, a glycoside of a compound having a phenolic OH group and a flavonoid-related compound can be produced. In addition, a glycoside by the α bond of hydroquinone, catechin and epigallocatechin could be produced with a glycosyl transfer rate of 20% or more by a usual batch reaction.

[Brief description of drawings]

FIG. 1 shows optimum pH and stable pH for hydrolysis by the present enzyme when soluble starch is used as a substrate. The vertical axis represents the relative activity when the hydrolysis activity of this enzyme at pH 7 is set to 100. The horizontal axis represents the reaction pH.

FIG. 2 shows the optimum pH and stable pH at which the present enzyme performs glycosyl transfer when soluble starch is used as a donor and hydroquinone is used as an acceptor. The vertical axis represents relative activity with the transglycosylation rate by the present enzyme at pH 5 as 100. The horizontal axis represents the reaction pH.

FIG. 3 shows the optimum temperature of the enzyme at pH 5.5,
And shows thermal stability. The vertical axis represents relative activity with the transglycosylation rate at 50 ° C. being 100. The horizontal axis shows the reaction temperature.

FIG. 4 shows the effect of this enzyme on various inhibitors. A solution containing 8 units of the enzyme (pH adjusted to 5.5) was added with 100 mM of the compound shown below,
After treatment at 1 ° C for 1 hour, the residual activity was measured according to the amylase activity measuring method.

FIG. 5 shows an ultraviolet absorption spectrum of this enzyme.
The vertical axis represents the absorbance. The horizontal axis represents wavelength.

FIG. 6 shows the amino acid composition of this enzyme.

FIG. 7 shows an elution curve of this enzyme by Superose 12 column chromatography. Vertical axis is 280n
The absorbance of m and the enzyme activity are shown. The horizontal axis shows the fraction number.

FIG. 8 shows a proton NMR analysis of hydroquinone glucoside obtained by the action of this enzyme.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Takahisa Nishimura, 2-18-1 -301, Showa-cho, Abeno-ku, Osaka-shi, Osaka (72) Inventor Hiroshi Takai, 1-chome, Nori, Nishi-yodogawa-ku, Osaka, Osaka 30-4 (72) ) Inventor Yoshinobu Terada 30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka

Claims (4)

[Claims] 1. A glycated α-amylase derived from a bacteria acts on a compound having a phenolic OH group or a flavonoid analogue and a glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches. A method for producing a glycoside, which comprises: 2. The bacterium is Bacillus.
It is a genus bacterium, The manufacturing method of the glycoside of Claim 1 characterized by the above-mentioned. 3. The bacterium is Bacillus subtilis (Ba).
Cylus subtilis) X-23 (Seikokuken Co., Ltd. Deposit No. P-13560), The method for producing a glycoside according to claim 1. 4. The method for producing a glycoside according to claim 1, wherein the bacterium is IFO14140.