JP2001046096A - PRODUCTION OF GLYCOSIDE WITH alpha-GLUCOSIDASE AND NEW alpha- GLUCOSIDASE AND ITS PRODUCTION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a conventionally unknown glycoside at a low cost, to produce an α-glucosidase capable of efficiently producing the glycoside and to provide a method for producing the α-glucosidase. SOLUTION: A transglycosylating reaction with an α-glucosidase can be utilized to thereby effectively produce an α-anomeric selective glycoside which is capsaicin, dihydrocapsaicin, nonylic acid vanillylamide catechin, epicatechin vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol or 5-nonyl alcohol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for efficiently producing glycosides using α-glucosidase, a novel α-glucosidase capable of efficiently performing a transglycosylation reaction with various receptors, and a method for producing the same. About.

[0002]

BACKGROUND ART An enzyme which hydrolyzes a compound having an α-glucoside bond from its non-reducing end to release α-D-glucose is α-glucosidase (EC 3.2.
1.20) and is present in various microorganisms, animals and plants. Enzymes classified as α-glucosidase include:
Not only the hydrolysis reaction, but also those capable of catalyzing the glycosyl transfer reaction together with the hydrolysis reaction are useful for the production of oligosaccharides and various glycosides.

For example, in the synthesis of oligosaccharides using α-glucosidase, a transglycosylation reaction using a sugar as an acceptor is used (J. Jpn. Soc. Starch S).
ci. , 35, 69-77 (1988)). On the other hand, regarding the synthesis of glycosides by α-glucosidase using a compound other than sugar as a receptor, “α-Glycosylflavones, their production methods and applications” (JP-A-4-3125)
No. 97), “Method for producing ethyl-α-glucoside”
(Japanese Patent Publication No. 6-30608).
-Method for producing menthyl-α-D-glucopyranoside ”
(Japanese Unexamined Patent Publication No. 9-224693), "Method for producing glycoside using immobilized enzyme" (Japanese Unexamined Patent Publication No. 9-313196), "Synthesis of ascorbic acid glycoside using α-glucosidase" (Chem. Pharm. Bull.,
38, 3020-3023 (1990)), "Enzymatic synthesis of glycosides" (J. Jpn. Soc. Starch).
Sci. , 35, 93-102 (1988)). However, methods for producing these glycosides are individually different due to enzyme substrate specificity, receptor specificity, and the like, and methods for efficiently producing various glycosides are still clearly evident. It has not been.

[0004] Capsaicin, dihydrocapsaicin,
It is the main component of the pungency of pepper and has been shown to exhibit various physiological activities. For example, it has been reported that β-adrenergic secretion is enhanced and the resulting increase in lipid metabolism and energy metabolism in adipose tissue, cholesterol-lowering effect, appetite enhancement, and salivary and gastric acid secretion are promoted (Japanese Journal of Nutrition and Food Science, 45). , 303-312 (19
92)). It is also known that nonyl acid vanillylamide is contained in a small amount in pepper, has a similar chemical structure to capsaicin, and exhibits the same physiological activity as capsaicin. Utilizing the various physiological activities of such capsaicins, they are expected to be applied to foods, cosmetics, and pharmaceuticals.However, they themselves have intense pungency and irritation, and their application fields and their usage are limited. I was In order to solve such a problem, reduction in pungency and irritation due to glycosylation of capsaicin has been studied (Japanese Patent Application Laid-Open Nos. 7-82289 and 10-25237). A method for producing glycosides of capsaicins is a method by chemical synthesis (J. Agric. FoodCh).
em. , 40, 2057-2059 (1992), JP-A-10-25237) and a method using plant cultured cells (JP-A-7-82289). The former requires expensive reagents and involves multiple steps. There is a problem that a reaction step is required, and in the latter case, the glycoside produced is limited to β-form, and there are problems such as a low yield. In addition, cyclodextrin glucosyltransferase (CGT) conventionally used for glycoside enzyme production.
ase), α-amylase, α-glucosidase, and a method for producing capsaicin glycoside using sucrose phosphorylase have not yet been reported.

Further, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol,
For glycosides of 3,4-dimethoxyphenol, tyrosinase inhibition (Biosci. Biotech.
Biochem. , 57, 1666-1669 (19)
93)), anti-mutagenic (Biosci. Biotec)
h. Biochem. , 57, 1290-1293.
(1993)). Regarding the production of the glycoside, CGTase (Biosc)
i. Biotech. Biochem. , 57,1
666-1669 (1993)), α-amylase (J. Ferment. Bioeng., 78, 3).
7-41 (1994)), sucrose phosphorylase (Biosci. Biotech. Bioche).
m. , 58, 38-42 (1994)), glycoside synthase (GSase) (Biosci. Biotech.
Biochem. , 58, 817-821 (199).
Although the synthesis using 4)) has been reported, these methods are inferior in that glycosylation efficiency is low, and in order to obtain a single glycoside because a plurality of glycosides are produced by the glycosylation reaction. There is a problem in that a complicated purification step is required, and α-
There is no report on a production method using glucosidase.

[0006] Furthermore, alkyl glucosides are used industrially as surfactants and synthetic intermediates for polymer materials. However, the production method thereof involves the production of glucose and various alcohols in an organic solvent having a low water content. There is a report that a yeast-derived α-glucosidase was used as a raw material for synthesis by a reverse reaction of a hydrolysis reaction instead of a sugar transfer reaction (Acta Alimentaria., 27, 2).
65-275 (1998)) alone was not satisfactory in terms of reaction efficiency, and a safety problem was considered in that an organic solvent was used in the reaction system.

On the other hand, the present inventors have already reported that Xanthomonas
That the glycosyltransfer reaction to menthol can be efficiently carried out by using microorganisms of the genera, Stenotrophomonas and Arthrobacter, and among them, Xanthomonas campestris WU-970 isolated by the present inventors.
1 (FERM BP-6578) was found to be the most excellent in terms of reaction efficiency (JP-A-11-155591).
No.). However, in Japanese Patent Application Laid-Open No. H11-155591, an enzyme that transfers glucose to menthol has not been isolated, and its properties have not been clarified. In addition, since the receptor specificity of carbohydrate-related enzymes such as α-glucosidase, α-amylase, and CGTase varies depending on the type of enzyme, the effect of this enzyme on receptors other than menthol was not clear.

[0008]

SUMMARY OF THE INVENTION The present invention has not been known so far in order to solve the problems that the above-mentioned prior arts have a very low reaction efficiency or require expensive reagents and multi-step reaction steps. It is an object of the present invention to provide a method for efficiently producing glycosides at low cost, a novel α-glucosidase capable of producing them efficiently, and a method for producing the same.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, by utilizing the transglycosylation reaction by α-glucosidase, capsaicin, dihydrocapsaicin, nonylate vanillylamide, It has been found that α-anomer-selective glycosides of catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol, and 5-nonyl alcohol can be efficiently produced. The present invention has been completed.

[0010] Furthermore, if α-glucosidase is an α-glucosidase having the following physicochemical properties, it has excellent glycosyltransferase activity with respect to the above-mentioned compounds, and can produce glycosides very easily and efficiently. Purify the enzyme,
After clarifying its properties, this enzyme is a novel α-
A glucosidase that can be obtained from a bacterium belonging to the genus Xanthomonas and a method for producing the enzyme have been found, and the present invention has been completed.

First, as an action, the α-1,4-bond of a saccharide is hydrolyzed, and glucose is transferred to an acceptor using maltose as a donor to synthesize a glycoside.

Next, maltose and p-nitrophenyl-α-D-glucopyranoside act as substrate specificities in the hydrolysis reaction, but maltotriose, maltohexaose, isomaltose, trehalose, sucrose,
Soluble starch, methyl-α-D-glucopyranoside, lactose, melibiose, cellobiose, amylose,
It has little effect on cyclodextrin, isomalttriose, nigerose, and kojibiose.

Receptor substrate specificity: menthol, ethanol, 1-propanol, 1-butanol, 2-butanol, isobutyl alcohol, 1-amyl alcohol,
Compounds having an alcoholic hydroxyl group such as 2-amyl alcohol and 5-nonyl alcohol, capsaicin, dihydrocapsaicin, nonylate vanillylamide, catechin,
Glycosyltransfer reaction can be performed by using a compound having a phenolic hydroxyl group such as epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol as an acceptor. The glycosides used are only monoglucosides.

It has a molecular weight of about 57,000 and has the following sequence as the N-terminal amino acid sequence. Sequence length: 20 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: N-terminal fragment

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Capsaicin, dihydrocapsaicin, vanillyl amide nonylate, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4 characterized by utilizing the transglycosylation reaction with α-glucosidase in the present invention In the method for producing an α-anomer-selective glycoside of dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol, or 5-nonyl alcohol, maltooligosaccharide or the like is used as a sugar used as a donor for the transglycosylation reaction. Preferably, maltose is used. As the reaction conditions, the donor is 5 to 60% (W /
V), preferably at a concentration of 15-40% (W / V), p
H5.0-9.0 solution, and capsaicin, dihydrocapsaicin, nonylate vanillylamide, catechin, epicatechin, vanillin, hydroquinone, catechol as a receptor for producing the glycoside of the present invention in the solution. Resorcinol, 3,4-dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol, or 5-nonyl alcohol is used in an amount of 0.01 to 10% (W / V), preferably 0.1 to 10% (W / V).
1 to 1% (W / V), and 0.01 to 10 units, preferably 0.1 to 1 unit of α-glucosidase in terms of maltose hydrolysis activity per 1 ml of the reaction solution, and then add 20 to 60 ° C. , Preferably at 30 to 50 ° C for 2 to 1
The reaction is carried out for 00 hours, preferably for 20 to 50 hours. still,
The method for measuring the maltose hydrolysis activity and the definition of the activity unit are described in Example 1. The method for recovering and purifying the glycoside thus produced from the reaction solution is performed by appropriately selecting or combining centrifugation, extraction with various solvents, various chromatography, recrystallization, etc. The glycoside of the invention can be produced.

In the above-mentioned glycosyltransfer reaction, as the α-glucosidase used in the present invention, a conventionally used α-glucosidase can be appropriately selected and used. In particular, α-glucosidase having the following physicochemical properties is used. -It is preferred to use glucosidases.

The present enzyme is an α-glucosidase identified for the first time in the present invention, and the glycoside can be produced very efficiently by using the present enzyme.

The physicochemical properties of this enzyme are as follows: (1) Action It hydrolyzes the α-1,4-bond of saccharide. Glucose is synthesized by transferring glucose to an acceptor using maltose as a donor.

(2) Substrate specificity of hydrolysis reaction 10 mM boric acid / potassium chloride containing 0.5% of substrate
Sodium hydroxide buffer (hereinafter referred to as borate buffer)
After reacting at 30 ° C. for 20 minutes, the glucose concentration in the reaction solution was measured using glucose CII-Test Wako. As shown in Table 1, this enzyme was found to be maltose and p-nitrophenyl- It acts on α-D-glucopyranoside, but has little effect on maltotriose, maltohexaose, isomaltose, trehalose, soluble starch, sucrose, lactose, methyl-α-D-glucopyranoside, melibiose and cellobiose. In addition, as a result of detecting the production of glucose by thin-layer chromatography, this enzyme was found to be amylose, cyclodextrin, isomalttriose, nigerose,
No effect on kojibiose.

[0020]

[Table 1]

(3) Receptor specificity of glycosyl transfer reaction Menthol, ethanol, 1-propanol, 1-butanol, 2-butanol, isobutyl alcohol, 1-
Compounds having an alcoholic hydroxyl group such as amyl alcohol, 2-amyl alcohol, and 5-nonyl alcohol, and capsaicin, dihydrocapsaicin, nonylate vanillylamide, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, and the like The compound having a phenolic hydroxyl group can efficiently undergo a glycosyl transfer reaction (Table 2). Glycosides that undergo glycosylation on these receptors and are produced are only monoglucosides.

[0022]

[Table 2]

(4) Optimum pH for Reaction As shown in FIG. 1, the optimum pH for hydrolyzing maltose is about pH 7 to 8, and as shown in FIG.
The optimum pH for saccharide transfer to menthol is about pH7.
0 to 8.5, and as shown in FIG. 3, the optimal pH for performing glycation to catechin is about pH 6 to 7, and FIG.
As shown in (1), the optimal pH for transglycosylation of epicatechin is about pH 5.5 to 6.5.

(5) Optimal temperature of glycosyltransfer reaction As shown in FIG.
The optimum temperature at H8.5 is 35 ° C. to 45 ° C., and as shown in FIG.
The optimum temperature at H6.5 is 40 ° C to 50 ° C, and as shown in Fig. 7, the optimum temperature at pH 6.0 when transglycosylating epicatechin is 35 ° C to 45 ° C.

(6) Effect of inhibitor As shown in Table 3, the glycosyltransfer activity of this enzyme is Cu²⁺, H
g²⁺Is almost completely inhibited by Zn²⁺, EDTA
To some extent by Mn²⁺, Mg ²⁺, C
a²⁺Have properties that are hardly inhibited.
won. The concentration of the inhibitor was 2 mM.

[0026]

[Table 3]

(7) Molecular weight As a result of SDS-polyacrylamide gel electrophoresis, the molecular weight was about 57,000.

(8) N-terminal amino acid sequence This enzyme has the following amino acid sequence at the N-terminal. Sequence length: 20 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: N-terminal fragment

In order to produce the enzyme of the present invention, that is, a novel α-glucosidase, as a strain to be used, a strain of the genus Xanthomonas described in Japanese Patent Application Laid-Open No. H11-155591 filed earlier by the present inventors was used. Bacteria, such as Xanthom
onas campestris WU-9701 (FERM BP
-6578) can be used. The medium to be used is not particularly limited as long as it is a medium generally used for culturing bacteria, but it is preferable to add a glucan having α-1,4 bond (maltose, starch, etc.) to the medium. For example, as a strain, Xanthomonas campestris W
When U-9701 (FERM BP-6578) is used, maltose 50 g / L, yeast extract
0 g / L, peptone 10 g / L, magnesium sulfate 7
A medium consisting of hydrate 1.0 g / L and adjusted to pH 7 can be used, and it is preferable to culture this at a culture temperature of 25 to 30 ° C. for 15 to 48 hours. Culture is performed aerobically while stirring or shaking. Next, since most of the present enzyme is present in the cells of the strain, the cells are collected from the culture of the strain by centrifugation or filtration. The obtained cells are subjected to a physical disruption method (fluid shear, solid shear, French press, sonication, freeze-thaw, etc.)
And a chemical destruction method (surfactant treatment, alkali treatment, enzyme treatment, etc.) are appropriately selected and destroyed singly or in combination. The crude enzyme, which is the α-glucosidase of the present invention, can be produced by purifying the cell lysate by removing insolubles by centrifugation or filtration. As a method for further purifying the enzyme from the obtained crude enzyme, a combination of ammonium sulfate salting out, centrifugation, dialysis, ultrafiltration, various types of chromatography, electrophoresis, etc., which are generally used as a method for purifying the enzyme, is used. The purified α-glucosidase of the present invention can be produced by performing these treatments.

[0030]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Method for Producing α-Glucosidase of the Present Invention Maltose 50 g / L, Peptone 10 g / L, Yeast Extract 2.0 g / L, Magnesium sulfate heptahydrate 1.0 g / L
Xanthomonas ca in medium adjusted to pH 7
mpestris WU-9701 was inoculated and cultured aerobically at 30 ° C. When the growth of the bacteria reached the stationary phase, the cells were recovered from the culture by centrifugation, and 10 mM citric acid-10
The cells were suspended in an mM phosphate buffer (hereinafter, referred to as citrate-phosphate buffer) (pH 7.0). The bacteria were disrupted by sonication, and the insoluble matter was removed by centrifugation to obtain a crude enzyme. Add 50 mg of ammonium sulfate powder to the obtained crude enzyme.
% Saturation and allowed to stand at 4 ° C. for 15 hours. The resulting precipitate was collected by centrifugation and 10 mM citric acid-
It was dissolved in a phosphate buffer (pH 7.0) and dialyzed against the same buffer. The obtained inner dialysis solution is applied to a DEAE-Toyopearl 650M (Tosoh) column equilibrated with the same buffer to adsorb the enzyme, and the sodium chloride concentration in the borate buffer is linearly increased from 0 to 0.5M. To elute. The active fraction was collected and dialyzed against borate buffer containing 0.1 M sodium chloride. The inner dialysis solution was applied to a DEAE-Toyopearl 650M (Tosoh) column equilibrated with the same buffer to adsorb the enzyme, and the sodium chloride concentration in the borate buffer was linearly increased from 0.1M to 0.3M. Elution was performed. The active fraction was collected, concentrated by ultrafiltration, applied to a Toyopearl HW-55 (Tosoh) column equilibrated with a borate buffer containing 0.15 M sodium chloride, and eluted with the same buffer. The active fraction was collected, concentrated by ultrafiltration, applied to a Sephacryl S-200 (Pharmacia) column equilibrated with a borate buffer containing 0.15 M sodium chloride, and eluted with the same buffer. The enzyme purified in this manner was purified 108 times in specific activity as compared with the crude enzyme, and was subjected to SDS-polyacrylamide electrophoresis to be purified into a substantially single band.

In the present invention, unless otherwise specified, as a method for measuring the enzyme activity of α-glucosidase, the method for measuring maltose hydrolysis activity is as follows: 0.2 ml of a 50 mM maltose aqueous solution, 50 mM boric acid / potassium chloride-water. 0.2 ml of sodium oxide buffer (hereinafter referred to as borate buffer) (pH 8.0) and 0.5 ml of distilled water are mixed, and after preincubation at 30 ° C., 0.1 ml of the enzyme solution is added to this solution. The reaction was performed at 30 ° C. for 10 minutes. The reaction was stopped by incubating the reaction in a boiling bath for 5 minutes. The concentration of glucose generated in the reaction solution by the hydrolysis reaction was measured using a commercially available glucose determination kit (Glucose CII-Test Wako, Wako Pure Chemical Industries, Ltd.), and 1 μmol of maltose was hydrolyzed in 1 minute to obtain 2 μmol of glucose. Is defined as one unit. Regarding the method for measuring glycosyltransferase activity, various receptors (0.3 to 1.0% in final concentration) were added to a buffer containing a predetermined concentration of maltose (the final concentration in the reaction system was set to 1.0 M). W / V)), the enzyme was added, and the mixture was shaken at a predetermined temperature for 24 hours. The amount of glycoside produced in the reaction solution was quantified by HPLC, and the transglycosylation activity was calculated from the amount produced as a relative activity.

Regarding the HPLC analysis method, HPLC analysis of menthol glycoside is described in Biosci. Bi
otech. Biochem. , 60 (11), 19
14-1915 (1996). The capsaicin glycoside and nonylate vanillylamide were also analyzed under the same conditions as for the menthol glycoside. HPLC of catechin glycosides and epicatechin glycosides
Analysis was performed using TSK-GEL ODS80-Ts (φ
4.6 × 250 mm, Tosoh), column temperature 40
And 20% methanol as a mobile phase at a flow rate of 1.0 ml /
min. HPLC analysis of oligosaccharides
sahipak NH2P-50 4E (φ4.6 × 2
50 mm, Showa Denko), column temperature 30 ° C., acetonitrile: 10 mM tetra-n-propylammonium = 7: 3 (V / V) as a mobile phase, flow rate 1.0 ml /
min.

Example 2 Glycosyltransferase Activity of α-Glucosidase of the Present Invention to Various Receptors and Method for Producing Various Glycosides of the Present Invention A borate buffer containing 1.0 M maltose (pH 8.
0) To 9 ml, add 50 mg of various receptors and 1 ml of the present enzyme having 10 units of maltose hydrolysis activity,
The mixture was shaken for 4 hours to react. The reaction was stopped by mixing 10 ml of the reaction solution and 40 ml of methanol, the insolubles were removed by centrifugation, and the supernatant was analyzed by thin layer chromatography to confirm the presence or absence of glycoside formation. TLC for thin layer chromatography
Aluminum sheet silica gel 60F ₂₅₄ (Merck)
And chloroform: methanol: water = 6: 4: 1 as a developing solvent. Spots were detected after spraying 10% sulfuric acid containing 2% cerium (IV) sulfate tetrahydrate,
The color was developed by heating on a hot plate. Table 2 shows the results of comparison of the amount of glycoside produced based on the spot size.

Glycoside production in the table is +++>++>+>
-Indicates that the amount of glycoside produced is large.

Thus, the capsaicin of the present invention,
Α-anomer selective glycoside of dihydrocapsaicin, nonylate vanillylamide, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol, 5-nonyl alcohol Was manufactured.

Example 3 Influence of pH on Glycosylation of Menthol by α-Glucosidase of the Present Invention A borate buffer containing 1.1 M maltose at a predetermined pH or an acid mixture-potassium hydroxide wide area buffer ( 0.1 g of menthol and 1 ml of the present enzyme having 10 units of maltose hydrolyzing activity were added to 10 ml of a wide area buffer (hereinafter referred to as a wide area buffer), and a 180 rpm reciprocal shaking reaction was performed at 40 ° C. for 24 hours. The amount of menthol glycoside produced was quantified by HPLC. The results are shown in FIG. The activity at the pH at which the amount of glycosides produced was the highest in each buffer was defined as 100 and expressed as a relative activity. As can be seen from FIG. 2, the optimum pH for menthol glycosylation by the present enzyme was pH 7.0 to 8.5.

Example 4 Effect of Temperature on Glycosylation of Menthol with α-Glucosidase of the Present Invention A borate buffer containing 1.1 M maltose (pH 8.
5) 0.1 g of menthol and 1 ml of the present enzyme having 10 units of maltose hydrolyzing activity were added to 10 ml, and a reciprocal shaking reaction at 180 rpm for 24 hours was performed at a predetermined temperature, and the amount of menthol glycoside produced was quantified by HPLC. FIG. 5 shows the results. The activity at 40 ° C., where the amount of glycosides produced was the largest, was expressed as a relative activity, taking the activity as 100. As can be seen from FIG. 5, the optimum temperature of menthol glycosylation by the present enzyme was 35 to 45 ° C.

[Example 5] Glycosylation of menthol with α-glucosidase of the present invention 10 mM borate buffer containing 1.0 M maltose (p
H8.0) was added to 10 ml of menthol, and 1.0 ml of the present enzyme having 10 units of maltose hydrolysis activity was shaken at 40 ° C. for 24 hours to react. Menthol in the reaction solution was completely disappeared due to glycosylation by the present enzyme, and formation of about 100 mg of menthol glycoside was confirmed.

Example 6 α-Glucosidase of the present invention
Of catechin by 10 mM citric acid-phosphate containing 1.0 M maltose
Add catechin and this enzyme to a buffer (pH 6.5), and add
The reaction was carried out by shaking at 24 ° C. for 24 hours. By extraction with ethyl acetate
After removing unreacted catechin from the reaction solution, the aqueous layer was separated into n-butane.
Extracted with ethanol. Butanol extract was added to Wakogel C
200 (Wako Pure Chemical Industries) column, elution solvent (n-porcine
Nol: 1-propanol: water = 10: 5: 4)
The solvent was distilled off from the fraction from which the reaction product eluted,
After washing with hexane, the solvent was completely removed to obtain the reaction product.
Was. The reaction product is dissolved in heavy water and 2,2-dimethyl-2
-Silapentane-5-sodium sulfonate (DSS)
A nuclear magnetic resonance (NMR) spectrum using
Specified. As a result, (+) catechin already reported
Chemical shift value of -α-glucoside (Biosci.
Biotech. Biochem. , 57 (10), 1
666 to 1669 (1993)).
Product identified as (+) catechin-α-glucoside
did. In FIG. ¹³The result of a C-NMR spectrum is shown.

Example 7 Effect of pH on Glycosylation of Catechin by α-Glucosidase of the Present Invention 60 mg of catechin was added to 10 ml of a citrate-phosphate buffer containing 1.2 M maltose at a predetermined pH, and maltose hydrolyzing activity was obtained. Add 50 mg of the enzyme having 5 units, and add
At 180 rpm for 24 hours. The amount of the produced catechin glycoside was quantified by HPLC. The results are shown in FIG. The activity at pH 6.5, at which the amount of glycosides produced was the largest, was expressed as a relative activity, taking 100 as the activity. As can be seen from Fig. 3, the optimal pH for catechin glycosylation by this enzyme
Had a pH of 6.0 to 7.0.

Example 8 Influence of Maltose Concentration on Glycosylation of Catechin by α-Glucosidase of the Present Invention 60 mg of catechin was added to 10 ml of 10 mM citrate-phosphate buffer at pH 6.5 containing a predetermined concentration of maltose, and maltose hydrolyzed. 50 mg of the present enzyme having 5 units of decomposition activity was added, and a 180 rpm reciprocal shaking reaction was performed at 45 ° C for 24 hours. The amount of the produced catechin glycoside was quantified by HPLC. FIG. 9 shows the results. The activity at a maltose concentration of 1.2 M where the amount of glycosides produced was the largest was expressed as a relative activity with 100 as the activity. As can be seen from FIG. 9, the optimal maltose concentration for catechin glycosylation by the present enzyme is 1.0 to 1.6.
M.

Example 9 Effect of Temperature on Glycosylation of Catechin by α-Glucosidase of the Present Invention 50 mg of catechin and 10 ml of maltose water were added to 10 ml of 10 mM citrate-phosphate buffer at pH 6.5 containing 1.2 M maltose. 50 mg of the present enzyme having 5 units of decomposition activity was added, and a reciprocal shaking reaction at 180 rpm for 24 hours was performed at a predetermined temperature, and the amount of catechin glycoside produced was quantified by HPLC. FIG. 6 shows the results. The activity at 45 ° C., where the amount of glycosides produced was the largest, was expressed as a relative activity, taking the activity as 100.
As can be seen from FIG. 6, the optimal temperature for epicatechin glycosylation by this enzyme was 40 to 50 ° C.

[Example 10] Under the optimum conditions, the α-
Glycosylation of catechin with glucosidase 60 mg of catechin and 50 mg of the present enzyme having 5 units of maltose hydrolyzing activity are added to 10 ml of 10 mM citrate-phosphate buffer (pH 6.5) containing 1.2 M maltose, and the mixture is added at 45 ° C. for 36 hours. The reaction was performed by shaking. As a result of quantifying the amount of catechin glycoside in the reaction solution by HPLC, 54.
06 mg of catechin glycoside was produced, and the yield was 5
It was 7.8% (mole conversion). In addition, as a result of HPLC analysis, there was only one peak considered to be derived from the generated catechin glycoside, and generation of a glycoside in which two or more molecules of glucose were bonded to catechin was not recognized.

Example 11 Effect of pH on Glycosylation of Epicatechin by α-Glucosidase of the Present Invention 50 mg of epicatechin, 10 mg of maltose and 10 ml of citrate-phosphate buffer containing 1.0 M maltose at a predetermined pH Add 50 mg of the present enzyme having 5 units of degrading activity and add 4 mg
A 180 rpm reciprocal shaking reaction was performed at 0 ° C. for 24 hours. The amount of produced epicatechin glycoside was quantified by HPLC.
FIG. 4 shows the results. PH at which glycosides were produced most
The activity in 6 was expressed as a relative activity with 100 as the activity. FIG.
As can be seen from the above, the optimal pH for epicatechin glycosylation by this enzyme was pH 5.5 to 6.5.

Example 12 Effect of Temperature on Glycosylation of Epicatechin by α-Glucosidase of the Present Invention 50 mg of epicatechin was added to 10 ml of 10 mM citrate-phosphate buffer (pH 6.0) containing 0.4 M maltose.
50 mg of the present enzyme having 5 units of maltose hydrolysis activity
Was added and the mixture was subjected to a 180 rpm reciprocal shaking reaction at a predetermined temperature for 24 hours, and the amount of produced epicatechin glycoside was quantified by HPLC. FIG. 7 shows the results. The activity at 40 ° C., where the amount of glycosides produced was the largest, was expressed as a relative activity, taking the activity as 100. As can be seen from FIG. 7, the optimal temperature for epicatechin glycosylation by this enzyme was 35 to 45 ° C.

Example 13 Glycosylation of Nonylate Vanillylamide with α-Glucosidase of the Present Invention 0.5 g of nonylate vanillylamide and 0.2 g of Twe
en80 was added to 5 ml of distilled water, and suspended while heating to 70 ° C. After adding and suspending 85 ml of 10 mM borate buffer (pH 8.0) containing 1.0 M maltose to this suspension, 10 ml of the present enzyme having 100 units of maltose hydrolysis activity was added, and the mixture was added at 40 ° C. for 40 hours. A shaking reaction was performed. After completion of the reaction, 400 ml of distilled water was added to the reaction solution, and extraction was performed twice with 500 ml of ethyl acetate. The ethyl acetate layer was collected, dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to dryness. The obtained extract was applied to a silica gel column, and eluted with chloroform: methanol = 7: 3 (V / V) to fractionate. The fraction eluted from nonylate vanillylamide glycoside was confirmed by thin-layer chromatography. The eluted fractions of nonylate vanillylamide glycoside were combined and the solvent was distilled off under reduced pressure to obtain 112 mg of nonylate vanillylamide glycoside. The yield at this time was about 15% in terms of mole. The obtained glycoside showed one peak by HPLC.

Example 14 Confirmation of the Structure of Nonylate Vanillylamide Glycoside Nonylate Vanillylamide Glycoside 1 prepared in Example 13
mg was suspended in 2 ml of 50 mM phosphate buffer (pH 6.8), and when yeast-derived α-glucosidase was allowed to act, glycosides were hydrolyzed to produce nonylate vanillylamide and glucose, and almond-derived Was not hydrolyzed by β-glucosidase. The amount of nonylate vanillylamide produced by the hydrolysis was quantified by HPLC analysis, and the amount of glucose was quantified by glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.). As a result, the molar ratio of the amount produced was glucose / nonylate vanillylamide = 1. It was confirmed that the resulting glycoside was a compound in which one molecule of nonylate vanillylamide was bound to one molecule of glucose as an α-anomer.

Example 15 Glycosylation of Capsaicin with α-Glucosidase of the Present Invention Glycosylation of capsaicin was performed in the same manner as in Example 13. The formation of capsaicin glycoside was confirmed by thin-layer chromatography analysis.

Example 16 Confirmation of the Structure of Capsaicin Glycoside When the capsaicin glycoside obtained in Example 15 was treated with α-glucosidase derived from yeast, it was hydrolyzed to produce capsaicin and glucose. , Was not hydrolyzed when treated with β-glucosidase derived from almonds. As a result of quantifying capsaicin and glucose produced from the capsaicin glycoside by α-glucosidase treatment in the same manner as in Example 14, the molar ratio of the produced amounts was glucose / capsaicin = 0.9. Further, the present glycoside was dissolved in deuterated methanol, and the NMR spectrum was measured.
-It was confirmed that this was a compound to which one molecule of glucose of the anomeric form was bonded. FIG. 10 shows the result of the ¹³ C-NMR spectrum.

[0051]

According to the present invention, an α-anomer-selective glycoside of capsaicin, dihydrocapsaicin, or nonylate vanillylamide, which has not been reported to be enzymatically synthesized, has been efficiently produced using α-glucosidase. . Further, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, propanol, butanol, isobutyl alcohol, amyl alcohol, and 5-nonyl alcohol are also efficiently used by using α-glucosidase to obtain α. Anomer-selective glycosides can be produced.

Further, the novel α-glucosidase of the present invention has an excellent glycosyltransfer reaction, and can produce glycosides very easily and efficiently. Therefore, it is useful for producing α-anomer-selective glycosides. It is. Furthermore, the present enzyme produces a mixture of glycosides in which one or several glucoses are bound to a receptor in the case of conventional α-glucosidase, but a glycoside in which one molecule of glucose is bound to the receptor by using the present enzyme. Only can be manufactured.

[Brief description of the drawings]

FIG. 1 is a characteristic diagram showing the relationship between the hydrolysis activity of maltose by the present enzyme and the reaction pH, wherein the vertical axis indicates the relative activity when the hydrolysis activity of the present enzyme at pH 7 is 100. The horizontal axis indicates the reaction pH.

FIG. 2 is a characteristic diagram showing the relationship between the glycosyltransferase activity to menthol and the reaction pH by the present enzyme, wherein the ordinate indicates the relative activity when the glycosyltransferase activity at the optimum pH of each buffer is set to 100. The horizontal axis shows the reaction pH.

[FIG. 3] Glycosyltransferase activity to catechin by this enzyme and reaction p
In the characteristic diagram showing the relationship of H, the vertical axis indicates the relative activity when the glycosyltransferase activity at pH 6.5 is 100, and the horizontal axis indicates the reaction pH.

FIG. 4 is a characteristic diagram showing a relationship between a glycosyl transfer activity to epicatechin by the present enzyme and a reaction pH.
The relative activity when the transglycosylation activity at 0 is 100 is shown, and the horizontal axis shows the reaction pH.

FIG. 5 is a characteristic diagram showing the relationship between the glycosyltransferase activity to menthol by the present enzyme and the reaction temperature, wherein the ordinate indicates the relative activity when the glycosyltransferase activity at 40 ° C. is set to 100. The horizontal axis indicates the reaction temperature.

FIG. 6 is a characteristic diagram showing the relationship between the glycosyltransferase activity to catechin by the present enzyme and the reaction temperature, wherein the ordinate indicates the relative activity when the glycosyltransferase activity at 45 ° C. is set to 100. The horizontal axis indicates the reaction temperature.

FIG. 7 is a characteristic diagram showing the relationship between the glycosyltransferase activity to epicatechin by the present enzyme and the reaction temperature, and the vertical axis indicates the relative activity when the glycosyltransferase activity at 40 ° C. is set to 100. The horizontal axis shows the reaction temperature.

FIG. 8: ^{13 of} catechin glycoside prepared using this enzyme
It is explanatory drawing which shows a C-NMR spectrum.

FIG. 9 is a characteristic diagram showing the relationship between glycosyltransferase activity to catechin and maltose concentration by the present enzyme, where the vertical axis indicates relative activity when the glycosyltransferase activity at a maltose concentration of 1.2 M is taken as 100. The abscissa indicates the maltose concentration.

FIG. 10 is an explanatory diagram showing a ¹³ C-NMR spectrum of capsaicin glycoside prepared using the present enzyme.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kotaro Kirimura 3-4-1 Okubo, Shinjuku-ku, Tokyo Waseda University Faculty of Science and Technology (72) Inventor Hiroyuki Nakagawa 3-4-1 Okubo, Shinjuku-ku, Tokyo Gakusei Waseda Within the Faculty of Engineering 3-2486 Machi (72) Inventor Yoshio Ito 3-26-11 Noshio, Kiyose-shi, Tokyo F-term (reference) 4B050 DD02 FF04E FF05E FF09E LL05 4B064 AF41 CA02 CA21 CB30 CC03 CD06 CD09 CD12 DA01 DA10

Claims (5)

[Claims] 1. Capsaicin, dihydrocapsaicin, nonylate vanillylamide, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol, 3,4-dimethoxyphenol, propanol characterized by utilizing a glycosyltransfer reaction by α-glucosidase. , Butanol, isobutyl alcohol, amyl alcohol, and 5-nonyl alcohol. 2. The α-glucosidase has the following physicochemical properties, ie, the action of hydrolyzing the α-1,4-bond of a saccharide and transferring glucose to an acceptor using maltose as a donor to synthesize a glycoside. Has and acts on maltose, p-nitrophenyl-α-D-glucopyranoside, but maltotriose, maltohexaose, isomaltose, trehalose, sucrose, soluble starch, methyl-α-D-glucopyranoside, lactose, Melibiose, cellobiose, amylose, cyclodextrin, isomalttriose, nigerose, has a substrate specificity of a hydrolysis reaction that hardly acts on kojibiose, and further has menthol, ethanol, 1-propanol, 1-butanol, 2-butanol, Isobutyl alcohol, 1-amyl alcohol Lumpur, 2-amyl alcohol, 5-compounds and capsaicin having an alcoholic hydroxyl group of the nonyl alcohol, dihydrocapsaicin, vanillylamide nonylate, catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol,
Glycosyl which has a substrate specificity of a receptor that performs a transglycosylation reaction using a compound having a phenolic hydroxyl group such as 3,4-dimethoxyphenol as a receptor, and is formed by performing a transglycosylation reaction with these receptors The method for producing a glycoside according to claim 1, wherein the body is a monoglucoside alone, has a molecular weight of about 57,000, and is an α-glucosidase having the following N-terminal amino acid sequence. Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment 3. α having the physicochemical properties according to claim 2.
-Glucosidase. 4. The α-glucosidase according to claim 3, which is produced by a bacterium belonging to the genus Xanthomonas. 5. A method for producing α-glucosidase, comprising culturing a bacterium belonging to the genus Xanthomonas and purifying the resulting culture.