JP2805273B2 - Glycoside production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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 analog and a glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches. There is no detailed study on the production of glycosides by the action of. For example, Amylase Symposium Vol, 101
975, pages 81-89, reports a study of transferring another saccharide to one saccharide and producing another saccharide. However, there is no study on an enzyme that performs glycosyl transfer to a phenol OH group in a compound and an OH group in a flavonoid analog. Further, it is not known that a compound having a phenolic OH group or a flavonoid-related compound becomes a glycoside by the action of a saccharifying amylase derived from bacteria.

[0003]

The compounds having a phenolic OH group used in the present invention include hydroquinone and dimethoxyphenol. Flavonoid analog compounds used in the present invention are flavone, isoflavone, flavonol,
Flavanone, flavanonol, catechin, auron, anthocyanidin, chalcone and dihydrochalcone. Bacterial saccharified α-amylases (hereinafter referred to as the present enzyme group) include, for example, Bacillus subtilis X-23 (Seikokenken No. P-13560) and IFO14140.
Glycated α-amylase (hereinafter referred to as amylase BSA) and the like. The method for producing the glycoside obtained according to the present invention is as follows. Compounds having a phenolic OH group such as hydroquinone or dimethoxyphenol, or flavone, isoflavone, flavonol, flavanone,
Glucan 1 having 1 to 20%, preferably 1 to 5%, of flavonoid analogs such as flavanonol, catechin, auron, anthocyanidin, chalcone or dihydrochalcone, and α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches The enzyme group of the present invention is added to a solution containing -20%, preferably 5-10%, and the mixture is allowed to act at a normal temperature and time to obtain a reaction solution. A purified sample 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. If no receptor such as a phenol analog or a flavonoid analog exists, it degrades (hydrolyzes) glucans having α-1,4 bonds, such as maltooligosaccharides, amylose, amylopectin, and various starches. However, if a phenol analog or a flavonoid analog and a glucan having α-1,4 bond such as maltooligosaccharide, amylose, amylopectin, various starches and the like are present at the same time, α-1 such as maltooligosaccharide, amylose, amylopectin, various starches and the like are present. Decomposes glucans having a, 4 bond and performs a sugar transfer to an OH group in a phenol analog or flavonoid analog by an α bond.

[0005] 2. Receptor specificity phenolic OH group or flavone in phenol analogs such as hydroquinone or dimethoxyphenol,
Glycosyl transfer is carried out by an α bond to an OH group in a flavonoid-related compound such as isoflavone, flavonol, flavanone, flavanonol, catechin, auron, anthocyanidin, chalcone or dihydrochalcone.

[0006] 3. Optimum pH and stable pH When hydrolyzing soluble starch, the optimal pH is pH 6
安定 7, and the stable pH is 5-8 (FIG. 1). When performing transglycosylation on hydroquinone, the optimal pH is pH 5
８8, and the stable pH is pH 4-13 (FIG. 2).

[0007] 4. Titration method (α-amylase activity measurement method) Amylase activity is measured as follows. 200 μl of 1.5% soluble starch (prepared to pH 5.5 with a 40 mM acetic acid-Na buffer solution) incubated at 40 ° C. was added to 50 μl of the enzyme solution incubated at 40 ° C.
Incubate at 0 ° C. for 10 minutes. The reaction is stopped by adding 2 ml of a 5: 1 mixture of 0.5N acetic acid and 0.5N hydrochloric acid to the reaction solution and stirring the mixture. This solution
Take 1 ml, add 5 ml of a solution containing 0.005% iodine and 0.05% potassium iodide and stir,
Leave at room temperature for 20 minutes. The absorbance at 660 nm of this solution is measured, and this absorbance is defined as C. Separately, a solution using purified water instead of the above enzyme solution is prepared as a blank, and the absorbance obtained by performing the same operation is referred to as B. Amylase activity A is obtained from C and B thus obtained by the following formula. A = (B−C) / B × 10 However, one unit of the amylase activity is assumed to be 10% of the B value.

(Measurement 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 are mixed at 1: 1.
React for 6-24 hours. The reaction solution was diluted 15-fold and HP
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 performed 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 equation. Glycosyl transfer rate = peak area of glycoside / peak area of control receptor x 100

[0009] 5. Range of suitable temperature for action Sugar transfer is stably performed from 30 ° C to 70 ° C at pH 5.5 (Fig. 3).

[0010] 6. Inhibitor Inhibited by copper ion and zinc ion, but hardly inhibited by EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion (FIG. 4).

7. Conditions for deactivation Completely deactivated when treated at 100 ° C. for 15 minutes.

8. Purification method After removing the 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
5.5 using a 0-0.5 M NaCl gradient) followed by dialysis. Next, phenyl sepharose column chromatography (using a 0.8M to 0M ammonium sulfate concentration gradient)
After dialysis, dialysis is performed. The dialysis solution is subjected to gel filtration using Superose 12 and freeze-dried to obtain a purified sample. The enzyme purified by such a method shows a single band by 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 Analysis of amino acid The amino acid composition is as shown in FIG.

Amylase BSA has the following physicochemical properties. 1. If no receptor such as a phenol analog or a flavonoid analog exists, it degrades (hydrolyzes) glucans having α-1,4 bonds, such as maltooligosaccharides, amylose, amylopectin, and various starches. However, if a phenol analog or a flavonoid analog and a glucan having α-1,4 bond such as maltooligosaccharide, amylose, amylopectin, various starches and the like are present at the same time, α-1 such as maltooligosaccharide, amylose, amylopectin, various starches and the like are present. Decomposes glucans having a, 4 bond and performs a sugar transfer to an OH group in a phenol analog or flavonoid analog by an α bond.

2. Receptor specificity To the phenolic OH group in phenol analogs such as hydroquinone or dimethoxyphenol, or the OH group in flavonoid analogs such as flavone, isoflavone, flavonol, flavanone, flavanonol, catechin, auron, anthocyanidin, chalcone or dihydrochalcone Carry out glycosyl transfer at α-linkage.

3. Optimum pH and stable pH When soluble starch is hydrolyzed, the optimum pH is pH
5.3, stable pH is 4.5-9.2
. When transglycosylating hydroquinone, the optimal pH is p
H6 and the stable pH is 5-8.

4. Titration method Same as the titration method for amylase X-23.

[5] Range of suitable temperature for action Sugar transfer is stably performed from 30 ° C to 60 ° C at pH 5.5.

6. Inhibitor Inhibited by copper ion and zinc ion, but hardly inhibited by EDTA, calcium ion, manganese ion, barium ion, magnesium ion and cobalt ion.

7. Conditions for deactivation Completely deactivated when treated at 100 ° C. for 15 minutes.

9. It has a molecular weight of about 47000.

[0024]

[Action] The action of the enzyme group of the present invention is as follows. 1. Degradation of starch The enzymes of the present invention hydrolyze starch to produce glucose, maltose, maltotriose and branched oligosaccharides. 2. 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 and maltose to phenolic OH groups and OH groups in flavonoid analogs. ,
Transforms maltotriose or the like by α-bonding.

[0025]

【Example】

(Example 1) Hydroquinone 10% and maltopentaose 20% were dissolved in a 20 mM acetate buffer (prepared to pH 5.5). Amylase activity 6 was added to 10 ml of this solution.
10 ml of an enzyme solution of amylase X-23 having 0 units was added. After allowing the above solution to react at 40 ° C. for 16 hours, a 3-fold amount of ethyl acetate was added and the mixture was vigorously shaken. After standing at room temperature for 30 minutes, the aqueous phase fraction was collected.
This operation was performed three times to completely remove unreacted hydroquinone, followed by drying under reduced pressure. The sample was dissolved in purified water, and the glycoside was adsorbed on an activated carbon column equilibrated with purified water. Next, unreacted sugar adsorbed on the activated carbon column was eluted with 20% methanol, and then the glycoside was eluted with 100% methanol. After the 100% methanol fraction was dried under reduced pressure, it was dissolved again in purified water and freeze-dried. Approximately 200 mg of hydroquinone glycoside
I got it. The purified sample was treated with α-glucosidase (manufactured by TOYOBO) and subjected to HPLC analysis as described in the above-mentioned method for measuring the glycosylation rate. Completely disappeared, and a new hydroquinone peak appeared. Thus, glucosyl hydroquinone is converted into hydroquinone with α
It was found to be a linked glycoside. Further, the structure of glucosyl hydroquinone was determined by NMR (JNM-GX270,
JEOL), and the same results as above were confirmed (FIG. 8). FIG. 8 shows the result of NMR analysis.
It was shown to. The transglycosylation rate due to this effect was about 27%. Further, 6 to 15 hours after the start of the reaction, the sugar transfer rate could be further increased by adding a new donor such as maltopentaose. Six hours after the start of the reaction, the sugar transfer rate when maltopentaose was added to 5% was about 40%.

Example 2 Hydroquinone 10% and maltopentaose 20% were added to a 20 mM acetate buffer (pH
5.5). To 10 ml of this solution,
Amylase BSA10 having 60 units of amylase activity
ml was added. After allowing the above solution to react at 40 ° C. for 16 hours, a 3-fold amount of ethyl acetate was added to the reaction solution, and the mixture was shaken vigorously. After standing at room temperature for 30 minutes, the aqueous phase fraction was collected. This operation was performed three times to completely remove unreacted hydroquinone, followed by drying under reduced pressure. The sample was dissolved in purified water, and the glycoside was adsorbed on an activated carbon column equilibrated with purified water. Then, unreacted sugar adsorbed on the activated carbon column was eluted with 20% methanol, and then eluted with 100% methanol.
The glycoside was eluted with% methanol. After the 100% methanol fraction was dried under reduced pressure, it was dissolved again in purified water and freeze-dried. By the above operation, about 150 mg of hydroquinone glycoside was obtained. This purified sample was treated with α-glucosidase (manufactured by TOYOBO) and subjected to HPLC as described in the method for measuring the glycosylation rate.
As a result of the analysis, the glucosylhydroquinone peak completely disappeared, and a new hydroquinone peak appeared. This revealed that glucosylhydroquinone was a glycoside in which glucose was α-bonded to hydroquinone. The sugar transfer rate by this action is about 22
%Met. In addition, 6 to 15 hours after the start of the reaction, a new donor, such as maltopentaose, was added to the reaction solution to further increase the sugar transfer rate. Six hours after the start of the reaction, the sugar transfer rate when maltopentaose was added to 5% was about 32%.

(Example 3) 2% of catechin and 10% of maltopentaose were dissolved in a 20 mM acetate buffer (prepared to pH 5.5). In 1 ml of this solution, 1 ml of an enzyme solution of amylase X-23 having 6 units of amylase activity was added.
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).
And eluted with a solution consisting of 12 parts 2 parts 86 parts of acetonitrile and ethyl acetate 0.05% phosphoric acid
Detected by nm), a new catechin glycoside peak was confirmed in addition to unreacted catechin. Further, regarding this reaction solution, α-glucosidase (TOY
OBO) and the same HPLC analysis was performed. As a result, the peak of the catechin glycoside disappeared and the peak of the unreacted catechin increased. From this, it was confirmed that the glycoside of catechin was a compound in which glucose was α-linked to catechin. In addition, the sugar transfer rate in this reaction was about 25%.

Example 4 2% of catechin and 10% of maltopentaose were dissolved in a 20 mM acetate buffer (prepared 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. The reaction solution was analyzed by HPLC (using an ODS column RP-18 and eluted with a solution consisting of 12 parts, 2 parts and 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid;
Detected by nm), a new catechin glycoside peak was confirmed in addition to unreacted catechin. Further, regarding this reaction solution, α-glucosidase (TOY
OBO) and the same HPLC analysis was performed. As a result, the peak of the catechin glycoside disappeared and the peak of the unreacted catechin increased. From this, it was confirmed that the glycoside of catechin was a compound in which glucose was α-linked to catechin. The sugar transfer rate in this reaction was about 22%.

Example 5 2% of epigallocatechin and 10% of maltopentaose were added to a 20 mM acetate buffer (pH
5.5). 1 ml of this solution contains 6
1 ml of an enzyme solution of amylase X-23 having a unit of amylase activity was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (using ODS column RP-18, acetonitrile, ethyl acetate and 0.1.
When the mixture was eluted with a solution consisting of 12 parts and 2 parts and 86 parts of 05% phosphoric acid and detected at 280 nm, a peak of a glycoside of epigallocatechin was newly confirmed in addition to unreacted epigallocatechin. Further, the reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of the glycoside of epigallocatechin disappeared and the peak of unreacted epigallocatechin increased. did. This confirmed that the glycoside of epigallocatechin was a compound in which glucose was α-linked to epigallocatechin. In addition, the sugar transfer rate in this reaction was about 28%.

Example 6 2% of epigallocatechin and 10% of maltopentaose were added to a 20 mM acetate buffer (pH
5.5). 1 ml of this solution contains 6
1 ml of an enzyme solution of amylase BSA having a unit of amylase activity was added and reacted at 40 ° C. for 16 hours. The reaction solution was 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 and 2 parts and 86 parts and detected by 280 nm.) In addition to unreacted epigallocatechin, a peak of a glycoside of epigallocatechin was newly confirmed. Further, regarding this reaction solution,
When a treatment with α-glucosidase (manufactured by TOYOBO) was performed and the same HPLC analysis was carried out, the peak of epigallocatechin glycoside disappeared and the peak of unreacted epigallocatechin increased. This confirmed that the glycoside of epigallocatechin was a compound in which glucose was α-linked to epigallocatechin. In addition, the sugar transfer rate in this reaction was about 25%.

Example 7 2% of epicatechin gallate and 10% of maltopentaose were added to a 20 mM acetate buffer (p
H5.5). In 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. The reaction solution was analyzed by HPLC (using an ODS column RP-18 and eluted with a solution consisting of 12 parts 2 parts 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. Further, the reaction mixture was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of epicatechin gallate glycoside disappeared and the peak of unreacted epicatechin gallate increased. did.
From this, it was confirmed that the glycoside of epicatechin gallate was a compound in which glucose was α-linked to epicatechin gallate. The sugar transfer rate in this reaction was about 1
3%.

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

Example 9 Epigallocatechin gallate 2
% And maltopentaose 10% 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 1 ml of the enzyme solution was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (using an ODS column RP-18 and eluted with a solution consisting of 12 parts 2 parts 86 parts of acetonitrile, ethyl acetate and 0.05% phosphoric acid and detected by 280 nm). In addition to epigallocatechin gallate in the reaction, a new glycoside peak of epigallocatechin gallate was confirmed. Further, regarding this reaction solution, α-glucosidase (TOY
When the same HPLC analysis was carried out by the treatment with OBO Co., Ltd., 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-linked to epigallocatechin gallate. In addition, the sugar transfer rate in this reaction was about 12%.

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

(Example 11) 2% of epicatechin and 10% of maltopentaose were added to a 20 mM acetate buffer (pH
5.5). 1 ml of this solution contains 6
1 ml of an enzyme solution of amylase X-23 having a unit of amylase activity was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (using ODS column RP-18, acetonitrile, ethyl acetate and 0.1.
When the mixture was eluted with a solution consisting of 12 parts and 2 parts and 86 parts of 05% phosphoric acid and detected at 280 nm), a peak of a glycoside of epicatechin was newly confirmed in addition to unreacted epicatechin. Further, the reaction mixture was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of epicatechin glycoside 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-linked to epicatechin. The sugar transfer rate in this reaction was about 23%
Met.

Example 12 2% of epicatechin and 10% of maltopentaose were added to a 20 mM acetate buffer (pH 5.
5). In 1 ml of this solution, an enzyme solution 1 of amylase BSA having 6 units of amylase activity was added.
The reaction was carried out at 40 ° C. for 16 hours.
The reaction solution was analyzed by HPLC (ODS column RP-
Using acetonitrile, ethyl acetate and 0.05%
When phosphoric acid was eluted with a solution consisting of 12 parts and 2 parts and 86 parts and detected by 280 nm), a peak of a glycoside of epicatechin was newly confirmed in addition to unreacted epicatechin. Further, the reaction mixture was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of epicatechin glycoside 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-linked to epicatechin. In addition, the sugar transfer rate in this reaction was about 23%.

(Example 13) 2% of 3.4 dimethoxyphenol and 10% of maltopentaose were dissolved in a 20 mM acetate buffer (prepared to pH 5.5). This solution 1
amylase having 6 units of amylase activity per ml
One ml of -23 enzyme solution was added, and the reaction was carried out at 40 ° C for 16 hours. The reaction solution was analyzed by HPLC (OD
It was eluted with 25% methanol using S column RP-18 and detected by 280 nm.
In addition to the 4-dimethoxyphenol, a new 3.4-dimethoxyphenol glycoside peak was confirmed. Further, the reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of the 3.4 dimethoxyphenol glycoside disappeared and unreacted 3.4 dimethoxyphenol was removed. 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) 2% of 3.5 dimethoxyphenol and 10% of maltopentaose were dissolved in a 20 mM acetate buffer (prepared to pH 5.5). This solution 1
amylase X having 6 units of amylase activity per ml
1 ml of the enzyme solution of -23 was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (O
Using a DS column RP-18, elution was carried out with 25% methanol and detection was performed at 280 nm). As a result, a new 3.5 dimethoxyphenol glycoside peak was confirmed in addition to unreacted 3.5 dimethoxyphenol. further,
About this reaction solution, α-glucosidase (TOYOB)
(Company O) and the same HPLC analysis was carried out. As a result, the peak of 3.5 dimethoxyphenol glycoside 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% of 2.3 dimethoxyphenol and 10% of maltopentaose were dissolved in a 20 mM acetate buffer (prepared to pH 5.5). This solution 1
amylase X having 6 units of amylase activity per ml
1 ml of the enzyme solution of -23 was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (O
Using a DS column RP-18, elution was carried out with 25% methanol and detection was performed at 280 nm). As a result, a peak of a glycoside of 2.3 dimethoxyphenol was newly confirmed in addition to unreacted 2.3 dimethoxyphenol. further,
About this reaction solution, α-glucosidase (TOYOB)
(Company O) and the same HPLC analysis was carried out. As a result, the peak of 2.3 dimethoxyphenol glycoside 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% of maltopentaose was dissolved in 20 mM acetate buffer (prepared 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 No. 3 was added and reacted at 40 ° C. for 16 hours. The reaction solution was analyzed by HPLC (ODS
Elute with 25% methanol using column RP-18.
(Detected at 80 nm), and aside from unreacted acetaminophenone, a new peak of a glycoside of acetaminophenone was confirmed. Further, the reaction solution was treated with α-glucosidase (manufactured by TOYOBO) and subjected to the same HPLC analysis. As a result, the peak of acetaminophenone glycoside disappeared and the peak of unreacted acetaminophenone increased. did. From this, it was confirmed that the glycoside of acetaminophenone was a compound in which glucose was α-bonded to acetaminophenone.

[0041]

As described above, glycosides of a compound having a phenolic OH group and a flavonoid analog can be produced by the enzyme group of the present invention. In addition, glycosides by α-linkage of hydroquinone, catechin and epigallocatechin could be produced at a sugar transfer rate of 20% or more by a normal batch reaction.

[Brief description of the drawings]

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

FIG. 2 shows the optimum pH and the 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 indicates the relative activity when the sugar transfer rate by this enzyme at pH 5 is 100. The horizontal axis indicates the reaction pH.

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

FIG. 4 shows the effect of the present enzyme on various inhibitors. In a solution containing 8 units of the present enzyme (pH is adjusted to 5.5), 100 mM of the following compound was added, and
After treatment at 1 ° C. for 1 hour, the residual activity was measured according to the amylase activity measurement method.

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

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

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

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

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroshi Takii 1-30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka (72) Inventor Yoshinobu Terada 1-30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka Examiner Hiroshi Taniguchi ( 58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 19/44 CA (STN)

Claims (4)

(57) [Claims] 1. A compound having only one phenolic OH group in a molecule or one of flavonoid analogous compounds having only one OH group in a molecule, and an α-type compound such as maltooligosaccharide, amylose, amylopectin and the like. A method for producing a glycoside, comprising reacting a glycated α-amylase derived from bacteria with a glucan having 1,4 bonds. 2. The method according to claim 1, wherein the bacterium is Bacillus.
The method for producing a glycoside according to claim 1, wherein the glycoside is a genus bacterium. 3. A compound having a phenolic OH group or a flavonoid analog and a glucan having an α-1,4 bond such as maltooligosaccharide, amylose, amylopectin or various starches, and Bacillus subtilis (Bacill).
(R. subtilis) X-23 (Seikoken-ken No. P-13560) -derived glycated α-amylase is allowed to act on the glycoside. 4. The method according to claim 1, wherein the bacterium is IFO14140.