KR101374748B1 - Method for producing catechin derivatives by using amylosucrase and catechin derivatives obtained therefrom - Google Patents

Abstract

The present invention relates to a method for producing a catechin derivative by an enzymatic reaction using catechin and low concentration of sucrose (sugar) as a substrate and amylosucrase as an enzyme, and a catechin derivative prepared therefrom. Catechin derivatives can be economically produced at high efficiency by enzymatic synthesis using less sucrose (sugar) as a substrate.

Description

Method for producing catechin derivatives using amylosucrase and catechin derivatives produced therefrom {Method for producing catechin derivatives by using amylosucrase and catechin derivatives obtained therefrom}

The present invention relates to a method for producing a catechin derivative using amylosucrase and to a catechin derivative prepared therefrom. More specifically, the present invention uses catechin and low concentration of sucrose as a substrate and amylosucrase as an enzyme. It relates to a method for producing a catechin derivative by an enzymatic reaction and to a catechin derivative prepared therefrom.

Catechin is an antibacterial substance and has anticancer effects. Research has also been shown to regulate whitening effects and cholesterol levels in mammals. However, catechins have limitations as various functional materials because they are not easily dissolved in water and are easily deteriorated by light. Sugar-transferred catechin product derivatives have proven to be more stable in UV light than conventional catechins. In addition, it was also found that the more the sugar is transferred than the conventional catechin, the more hydrophilic. Thus, catechin sugar transition product derivatives can be useful in many ways.

Regarding catechins, Korean Patent No. 10-2000-0055727, 'Methods for Preparing Tannins Derived from Plants and Derivatives thereof,' describes a method for obtaining catechins from Yulpi using an organic solvent. 2003-0070710 'Catechin derivative' describes a method for obtaining a catechin derivative by introducing another functional group instead of a gallate group in oxygen of catechin, and a Korean Patent Application No. 10-2008-7000724 'New Fatigue' Catechin derivatives describe pyrocatechins which are used as useful intermediates in the preparation of medicinal active ingredients. In addition, the Republic of Korea Patent Application No. 10-2005-0117086 'method of preparing a derivative of sugar-transfer compounds using sugar transfer enzymes and derivatives prepared therein' is a saccharide transfer of catechin or the like using glucan sucrase or levane sucrase A method for synthesizing derivatives of compounds is described, and in the art, the synthesis efficiency is low in the case of derivative synthesis by dextran sucrase, and the synthesis efficiency is relatively high in the case of derivative synthesis by sucrose phosphatase, but 30% There is a known problem that requires a high concentration of sucrose.

The present invention is to develop a method for producing catechin derivatives that have overcome the disadvantages of catechins by using an catechin as a substrate and a relatively cheap sucrose at a low concentration and an enzyme reaction using amylosucrase. The purpose is to provide.

In order to achieve the above object, the present invention provides a method for producing a catechin derivative, characterized in that the addition of amylosucrase enzyme to the reaction solution containing sucrose (sugar) and catechin as a substrate.

Amylosucrase (ASase) is a glycosyltransferase belonging to glycosyl hydrolase family 13 (GH13). This enzyme promotes hydrolysis of glycoside bonds in sucrose to generate glucose and fructose, as well as to synthesize α-glucan polymers by reaction with the generated glucose molecules. Recently, amylosucrases have been found to synthesize a variety of glycosyl transfer products using various biocompatible materials in addition to glucose as receptor molecules. In addition, Neisseria processed by semirational engineering Amylosucrase from polysaccharea allowed site-specific glucosylation of non-natural receptors.

The amylosucrase used in the present invention is not particularly limited in its origin, but is preferably Deinococcus amylosucrase (hereinafter referred to as 'DGAS') derived from geothermalis ). Deinococcus Amylosucrase derived from geothermalis ) can overcome the disadvantages of the sugar transfer reaction of the existing sugar transfer enzymes dextran sucrase, sucrose phosphatase. That is, it has the advantage of synthesizing catechin derivatives with high efficiency even using a small amount of sucrose.

On the other hand, Dinococcus zeomales of the present invention (Deinococcus geothermalisAmylosucrase derived from) may preferably be used in which the tag for purification is bound to the N-terminus or C-terminus.

Typically, the enzyme is obtained from the transformed transformed by introducing the gene encoding the enzyme, it must be purified to recover the enzyme. In the purification process, it is very important to increase the purity of the enzyme, and a method of tagging 5 'or 3' of the gene for purification is generally used. As a tag for purification, various ones can be selected in consideration of the pH characteristics of host proteins included in the strain used as a host. When E. coli is used as a host, cationic is considered when considering the pH of the main protein in E. coli. It may be efficient to use His-tag, an amino acid tag.

On the other hand, the activity of the enzyme is often changed by tagging, but Deinococcus geothermalis ) -derived amylosucrase showed no significant change in activity even with a heat-tag (His-tag).

In addition, the present invention provides a catechin derivative represented by the following formula (1) by the above method:

Wherein R is

to be.

The catechin derivative manufacturing method of the present invention can economically produce catechin derivatives at high efficiency by enzymatic synthesis method using less sucrose as a substrate than a conventional substrate.

Figure 1 shows the glycosyl transfer activity of DGAS in the mixture by TLC analysis, where M is a G1 to G7 standard, Su is 1% sucrose and F is 1% fructose. In addition, C represents a catechin glycosyl transfer reaction without an enzyme, R represents a catechin glycosyl transfer reaction of DGAS using an enzyme, CT1 represents a catechin glucoside, and CT2 represents a catechin maltoside.
Figure 2 shows an HPLC analysis of the glycosyl transfer reaction of DGAS by catechin and sucrose, where A is the glycosyl transfer reaction of DGAS with 1: 1 mole ratio of catechin and sucrose, and B is at a 1:10 molar ratio. Glycosyl transfer reactions with catechin and sucrose are shown on the chromatogram, 1 for catechin, 2 for catechin glucoside (CT1), and 3 for catechin maltoside (CT2).
3 shows TLC analysis of glycosylation transfer products by various enzymes, where M is a G1 to G7 standard, Su is 1% sucrose, F is 1% fructose, and lane 1 is Glycosyl transfer reaction of DGAS with catechin and sucrose at 1: 1 molar ratio, lane 1 is treated with α-glycosidase and lane 3 is treated with β-glucosidase. , Lane 4 is a lane 1 sample treated with LGMA, lane 5 shows a glycosyl transfer reaction of DGAS with catechin and sucrose in a 1:10 molar ratio, lane 6 is a glycosidase of lane 5 Lane 7 is lane 5 sample treated with β-glucosidase, lane 8 is lane 5 sample treated with LGMA, CT1 is catechin glucoside, CT2 is catechin maltoside, CT3 is catechin maltose Triosai A.
Figure 4 schematically shows the α-glucosidase, β-glucosidase and LGMA enzyme assays for the catechin glycosyl transfer reaction of DGAS and the structural analysis of catechin glucoside and catechin maltoside.
FIG. 5 shows TLC and HPLC analysis of purified catechin glucoside and catechin maltoside, where M is 1% sucrose and 1% fructose, C is catechin, CT1 is catechin glucoside, and CT2 is Catechin maltoside.
6 is a preliminary cyclic HPLC and TLC analysis of the catechin glycosyl transfer reaction of DGAS for catechin maltooligosaccharide separation, where 1 represents catechin, 2 represents catechin glucoside, 3 represents catechin maltoside, 4 represents catechin maltotrioside, 5 represents catechin maltotetraside, and 6 represents catechin maltopentaoside.
FIG. 7 shows catechin glycosyl transfer reaction by DGAS with different receptor ratios for donors, S and F in lane 1 show sucrose and fructose standards, respectively, and lane 2 shows control response. Lane 1 shows the reaction using a 1: 1 molar ratio of receptor to donor ratio, lane 4 shows the reaction using a 1: 5 molar ratio of receptor to donor ratio, and lane 5 shows a 1:10 molar ratio of receptor to donor ratio. Reaction using ratio is shown.

The present invention will be described in more detail with reference to the following Examples, which are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Compounds and Enzymes

Sucrose and catechin hydrates were purchased from Duchefa Biochemistry (Hahalem, Netherlands) and Sigma-Aldrich (St. Louis, Missouri, USA). To determine glycoside binding of glycosyl transfer products, yeast α-glucosidase (OYC Americas, Andover, Mass.), Sulfolobus shibatae β-glucosidase and Lactobacillus gasseri maltose Various glycoside hydrolases were used, including genomic amylase (LGMA). All other compounds used in the present invention were reagent grade for analysis.

Strain and culture conditions

Escherichia coli BL21 [F-, omp T, hsd SB (rB-, mB-), dcm, gal, (DE3)] containing dgas ( D. geothermalis amylosucrase ) cloned into pGEX vector (pGEX-DGAS) ) Was used for DGAS production in this example. Luria-Bertani (LB) liquid medium containing 1% tryptone (w / v), 0.5% yeast extract (w / v) and 1% sodium chloride (w / v) with ampicillin (100 μg / mL) broth medium) was used for proliferation of E. coli BL21 cells.

Preparation of Recombinant DGAS

Recombinant E. coli BL21 containing pGEX-DGAS cells were grown in 500 mL LB medium with 0.1 mg / mL ampicillin at 37 ° C. for 3 hours to obtain 0.5-0.6 optical density at 600 nm. A final concentration of 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was used for induction. After induction, cells were further incubated at 18 ° C. for 14 hours. Induced cells were harvested by centrifugation at 10,000 × g for 10 minutes. The pellet was resuspended in phosphate salt buffer (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ and 1.8 mM KH ₂ PO ₄ , pH 7.4) and resuspended in ultrasound (Sonifier 450, Branson, Connecticut, USA). Danbury material: output 4, 6 times in 30 seconds, constant duty) cells were disrupted (disrupted). Finally, the extracted protein in the solution was collected by centrifugation at 15,000 xg for 20 minutes.

Glutathione-Sepharose® high performance affinity chromatography using a 0.8 x 4 cm affinity chromatography column (Bio-RAD, Hercules, CA, USA), GE Healthcare Bio-Science AB, Recombinant DGAS was purified using Uppsala, Sweden. Tris-HCl buffer (pH 8.0) containing reduced glutathione 30 mM was used for purification. Specific purification steps are described above. The harvested recombinant DGAS was dialyzed by dialysis seamless cellulose test tube (Sigma-Aldrich) with optimal buffer (50 mM Tris-HCl, pH 7.0). After dialysis, purified DGAS was concentrated on a Vivaspin column (30,000 MWCO; Sartorius Stedim Biotech. GmbH, Goettingen, Germany) using centrifugation until the final protein concentration reached 12 mg / mL in optimal buffer.

Determination of enzymes and their activity

Cell extracts and purified enzymes were identified by SDS-PAGE. A 10% polyacrylamide gel was prepared for SDS-PAGE analysis. 5 μl aliquots of each sample were transferred to a new microcentrifuge tube, and 3 μl of 3 × loading dye was added thereto. The samples were mixed, boiled for 2 minutes and then loaded onto a polyacrylamide gel. The gel was run for 45 minutes at 200 V / 6.5 cm and stained using Coomassie Brilliant Blue R-250.

DGAS activity analysis was performed using 146 mM sucrose and enzyme in 50 mM Tris-HCl pH 7.0 at 45 ° C. for 10 minutes. After the enzymatic reaction, the resulting fructose was measured using the 3,5-dinitrosalicylic acid (DNS) method. Fructose was used as the standard to calculate the fructose concentration in the reaction mixture. Inactivity was defined as the amount of enzyme required to hydrolyze sucrose relative to 1 μmol fructose per minute under reaction conditions.

Biosynthesis of Catechin Glycosides

Glycosyl transfer reactions on catechins were carried out using three concentrations of sucrose. The basic reaction mixture for glycosyl transfer consisted of 50 mM DGAS per mg of sucrose in receptor catechin 25 mM, donor 25 mM sucrose, and 50 mM Tris-HCl buffer, pH 7.0. The catechin concentration was fixed at 25 mM, the final concentration in the reaction mixture, while the sucrose concentration was changed. The three molar ratios between catechin and sucrose in this example were 1: 1, 1: 5 and 1:10 (catechin: sucrose). To produce the catechin glycoside product, a glycosyl transfer reaction was conducted in 50 mM Tris-HCl buffer (pH 7.0) at 30 ° C. instead of 45 ° C. The glycosyl transfer activity of DGAS was higher at 30 ° C. than at 45 ° C. Because it is known.

Thin layer chromatography ( TLC ) And high performance liquid chromatography ( HPLC ) analysis

TLC and HPLC analysis were performed to detect glycosyl transition products. For TLC analysis, TLC silica gel plate 60F ₂₅₄ (Merck, Whitehouse Station, NJ) was activated at 110 ° C. for 30 minutes and ethyl acetate: acetic acid: water (3: 1: 1, v: v: A separation solvent consisting of v) was used. Samples were expressed on TLC plates after the TLC plates were completely dried in an oven. The origin of the catechin glycoside was defined by visualization under a Reprostar 3UV detector (CAMAC, Muttenz, Switzerland) at 280 nm. Finally, after rapidly immersing the TLC plate in 0.3% (W / V) N- (1-naphthyl) -ethylenediamine and 5% (v / v) H ₂ SO ₄ in methanol, the visible point is clearly observed. Dry in an oven at 110 ° C. until.

For HPLC analysis, the reaction mixture is centrifuged, filtered through a 0.2 μm nylon 66 syringe filter (Whatman, Kent, UK), a Shimadzu LC10 pump and Shimadzu SPD-10A UV detector (Shimadzu, Tokyo, Japan) at 280 nm Analysis was performed by Luna 5μ NH ₂ (Phenomenex, Torrance, Calif., USA) connected to a Shimadzu model SCL-10 system equipped with a. The product was isolated using a mobile phase solvent consisting of acetonitrile and water and 1% phosphoric acid (7: 3, v / v) at a flow rate of 1.0 mL / min.

Open column  Chromatography ( open column chromatography ) And Glycosyl  Recirculation for Separation of Transitioned Products HPLC

While catechin glycosyl transfer products were prepared on a large scale using open column chromatography, small amounts of small amounts of glycosyl transfer products were obtained using recycle-HPLC. The stationary phase used was silica gel 60 (Merck) powder and the mobile phase solvent consisted of ethyl acetate: methanol: water (40: 4: 1, v / v / v). The reaction mixture sample was dried over silica gel powder on a rotary evaporator (EYELA, Tokyo, Japan). The fully dried sample was loaded flat onto silica gel (3.5 cm diameter, 12 cm height) in an open column and the flow rate of the mobile phase was 1.5 mL per minute. Each fraction (10 mL) was collected in vitro and separation of glycosyl transfer products was confirmed by TLC analysis. Compounds separated in the eluate were dried using a lyophilizer (Il-shin Lab, Suwon, Korea) and collected for further analysis.

A preparative HPLC system for recycling (LC-9104, JAI, Tokyo, Japan) was used for the small percentage of catechin glycoside separation. 3 mL of sample was applied to JAIGEL-W251 (2 cm x 50 cm, JAI) simultaneously connected to a JAIGEL-W251 (2 cm x 50 cm, JAI) and guard column. The sample was eluted with deionized water at 1.8 mL / min flow rate. Fractions corresponding to the detected peaks were collected and lyophilized. The purity of each sample was confirmed by TLC analysis.

Glycosyl  Of transition products Enzymatic  Structure Determination Based on Hydrolysis

The structures of various catechin glycosides were derived based on enzymatic degradation by α-glucosidase, β-glucosidase and maltogenic amylase. The catechin glycosyl transfer reaction mixture was further reacted with three enzymes at the optimum reaction conditions of each enzyme. The α-glucosidase reaction was carried out at 37 ° C., while the β-glucosidase reaction was carried out at 90 ° C. The reaction temperature of maltogenic amylase (LGMA) was 50 ° C.

NMR  analysis

¹ H and ¹³ C NMR spectra of catechin and purified catechin glycosides were obtained by Varian Inova AS 400 MHz NMR spectrometer (Varian, Palo Alto, CA, USA). Samples were dissolved in DMSO- d6 at 24 ° C. and tetramethylsilane (TMS) was used as a chemical shift reference.

As a donor molecule Sucrose  Used DGAS By catechins Glycosyl  transition

As shown in FIG. 1, catechins can be successfully used as receptors for the glycosyl transfer activity of DGAS. Two points (CT1, CT2) were shown in TLC analysis (FIG. 1). These points were also detected in UV light, meaning that catechins are derived from catechins in that they are the only fluorescent compounds in the reaction. In addition, HPLC analysis confirmed the presence of two glycosyl transfer reaction products of catechin (FIG. 2). When glycosyl transfer of salicycin using amylosucrase from N. polysaccharea , two salicycin glycoside transition products were detected in TLC and HPLC analysis. These salicylic glycoside shift products have been found to be glucosyl-salicyl and maltosyl-salicyine. Likewise, the two glycosylated catechin products observed in the DGAS glycosyl transfer reaction appear to be glucosyl-catechin and maltosyl-catechin.

Possible structures of two glycosylated catechin products were determined by enzymatic analysis (FIG. 3). Three glycoside hydrolases, including α-glucosidase, β-glucosidase and maltogenic amylase, were used to hydrolyze the DGAS reaction product of the catechin. α-glucosidase (EC 3.2.1.20) acted on α-1,4-glycosidic bonds in many α-glucoside substrates to produce glucose. β-glucosidase catalyzes the hydrolysis of β-1,4-glycosidic bonds in β-linked glucoside molecules, resulting in glucose. Unlike these enzymes, maltogenic amylases hydrolyze α-1,4-glycosidic bonds similar to α-glucosidase, but produce maltose instead of glucose.

After the α-glucosidase reaction, spots corresponding to glucose appeared, while those corresponding to compound CT1 were no longer present (FIGS. 3, 2). There was no change in point in the β-glucosidase response (Figures 3, 3 lanes). In contrast, although the compound CT2 was not produced by the catalytic action of LGMA, it was found to correspond to maltose (lanes 3 and 4).

These results indicate that glucose molecules produced by DGAS hydrolysis were attached to catechins via α-1,4-glycoside bonds by the glycosyl transfer activity of DGAS. Compound CT1 is also believed to be glucosyl-catechin, where one glucose molecule is linked to catechin. Compound CT2 appears to be maltosyl-catechin, where two glucose molecules are linked to catechin.

Interestingly, CT2 points did not disappear completely in the presence of α-glucosidase, but CT1 points did not occur at all. This situation is believed to be due to the greater substrate specificity of α-glucosidase for maltose than for maltotriose. Likewise, CT1 can be much more substrate friendly than CT2, so that a small amount of CT2 may remain after reaction with α-glucosidase.

Isolation and Structural Analysis of Catechin Glycosides

To determine the correct chemical structure, the two compounds shown in TLC analysis (CT1 and CT2) were purified using open column chromatography as described above. Two catechin glycosyl transfer products were successfully separated as shown in FIG. 5 and their structures were determined using NMR analysis: ¹ H NMR and ¹³ C NMR results are shown in Table 1.

The molecular weight of CT1 was calculated to be 452 daltons according to FAB mass spectrometry. These results indicate that CT1 is catechin monoglucoside, where one molecule of glucose is attached to catechin. ¹ H NMR and ¹³ C NMR confirmed that the structure of CT1 was catechin monoglucoside (Table 1). The binding site of glucose was C-3 'from the results of ¹ H NMR, and according to ¹³ C NMR results, the chemical shift of C-3' (146.1) was basically unchanged and the chemical shift of H-2 '(δ7.29 ) Is lower than in free catechins. The α-batch form of the anomer carbon in glucose was found by the coupling constant (J = 3.6 Hz) in the ¹ H NMR spectrum. Thus, the structure of the CT1 compound was determined to be catechin-3'-0-α-D-glucopyranoside, wherein the glucose molecule was linked to the C3 'position of the catechin.

The molecular weight of compound CT2 was determined to be 614 Daltons because two peaks appear in m / z 615 ([M + H] ⁺ ) and m / z 637 ([M + H] ⁺ ) in FAB mass spectrometry. Like the structure of CT1, the chemical structure of CT2 was confirmed by ¹ H NMR and ¹³ C NMR. All of these NMR spectra showed that the molar ratio of catechin to glucose in the CT2 compound was 1: 2 (see Table 1). In addition, the bond between the two glucose molecules was found to be α-1,4-glycosidic binding according to the coupling constant (J = 3.6 Hz) in the ¹ H NMR spectrum. By this result, the structure of compound CT2 was concluded to be catechin-3'-O-α-D-maltoside, wherein two glucose molecules (maltose) were linked to the C3 'position of the catechin. The structure of these two catechin glycosyl transfer products exactly matched the structure of the expected compound obtained by enzymatic analysis (FIGS. 3 and 4).

In the above, the glycosyl transfer mechanism of amylosucrase appears to transfer glucose molecules resulting from the hydrolysis of sucrose to sugar residues of various receptor molecules. This transition has been reported in the formation of amylose-like polymers, salicylic acid, and arbutin-glycosyl transition products. As a result, an α-1,4-glycosidic bond was formed between the transitioned glucose and the sugar residue in the receptor molecule. The phenolic aglycone compound may also act as a receptor molecule of amylosucrase glycosyl transfer activity.

Reaction conditions for the production of catechin glycosyl transition products

Various reaction conditions were studied to obtain the maximum yield of catechin glycosyl transfer products. First, the reaction temperature was studied in the 25 to 45 ℃ temperature range. At higher temperatures (40-45 ° C.), the yield of catechin glycosyl transition products from DGAS was significantly lower than at 35 ° C. as expected based on the DGAS characteristics. DGAS showed various dual reactions (hydrolysis and glycosyl transition) depending on the reaction temperature. Glycosyl transitions preferably occur at lower temperatures, while hydrolysis occurs mainly at higher temperatures. In general, glycoside hydrolases not only create, but destroy, glycosidic bonds in the substrate molecule according to thermodynamic equilibrium. Hydrolysis reactions that break down glycoside bonds by glycoside hydrolases are more advantageous at higher temperatures until unfolding temperatures are reached. However, under suitable conditions such as optimal water content, BSA addition, the presence of organic solvents and high receptor concentrations, the hydrolase can be induced to form glycosides. However, the reaction temperature may be lower than 35 ° C. in this example. At temperatures lower than 30 ° C., the solubility of catechins decreases to produce a white catechin precipitate. Therefore, the reaction temperature of DGAS glycosyl transition of catechin was 35 degreeC.

The effect of the ratio of receptor and donor concentrations was studied. The reaction products were studied using two ratios, 1: 1 and 1:10 (receptor: donor, molar basis) and using TLC and HPLC analysis. The concentration of catechin was fixed at 25 mM due to low solubility. At a 1: 1 ratio, TLC analysis showed that CT1 was the major glycosyl transition product and there was a relatively small amount of CT2 (Figure 3, lane 1). However, at the 1:10 ratio, the CT1 points disappeared, with most of the points corresponding to CT2 (Figures 3, 5 lanes). Interestingly, some smear was observed above the fructose point, indicating that there were some other glycosyl transition products synthesized by DGAS.

The presence of catechin glycosyl transfer products in addition to CT1 and CT2 was confirmed by recycle preparative HPLC analysis (FIG. 6). Peaks appearing in the HPLC chromatogram showed regular intervals between the peaks. In addition, these compounds are clearly derived from catechins based on UV as the detection method. Thus, the peak is believed to correspond to the various catechin maltooligosaccharides to which maltooligosaccharides (maltotriose, maltotetraose and maltopentao) are attached to the catechin molecule. Enzymatic degradation of these compounds by maltogenic amylase produced catechin and catechin-3′-O-α-D-glucopyranoside spots according to TLC analysis. Thus, DGAS appears to produce predominantly catechin-3′-O-α-D-glucopyranoside points at low donor-to-receptor ratios (1: 1). As the donor concentration increased, relatively more catechin-3′-O-α-D-maltosides and other catechin maltooligosaccharides were synthesized (FIG. 7). This means that DGAS transfers glucose residues resulting from sucrose hydrolysis to another glucose molecule. The obtained product, catechin-3'-O-α-D-glucopyranoside, can be used as a receptor when produced in an appropriate amount. These results indicate that the profile of glycosyl transfer products can be controlled by adjusting the ratio of receptors to donor molecules in the DGAS reaction. Thus, glycosyl transition products can be selectively synthesized to simplify separation.

The effect of enzyme concentration on the DGAS glycosyl transfer reaction was studied. The higher the enzyme (DGAS) concentration used in the reaction, the faster the glycosyl transfer reaction rate. Increasing the DGAS concentration from 50 U / mL to 500 U / mL shortened the final reaction time, resulting in an equivalent amount of glycosyl transfer reaction product in just 1 hour rather than 24 hours. However, the profile of the catechin-glycosyl transfer product did not change at different concentrations of the enzyme.

In the above, several carbohydrate-active enzymes have been reported to produce catechin glycosides. Trichoderma α-amylase from viride JCM22452 was able to glucosylate a wide range of natural flavonoids, especially catechins. The glucose donor molecule of this reaction was dextrin. The catechin glycosides synthesized vary and their structures are catechin-5-O-α-D-glucopyranoside, catechin-5-O-α-D-maltoside and catechin-4'-O-α- It was determined to be D-maltoside. Cellulase from Aspergillus niger was used to synthesize catechin-β-D-fucopyranoside using p-nitrophenyl-β-D-fucopyranoside as donor molecule. A brush as Bacillus stearate Russ (Bacillus α-glucosidase from stearothermophilus ) is catechin-7-O-α-D-glucopyranoside and catechin-5-O-α-D-glucoid present in approximately equal amounts using maltose as donor molecule. It is possible to produce a mixture of ranosides. The chemical structural formulas of these obtained products are different from those of the glycosyl transition products (CT1 and CT2) found in the DGAS reaction. These results indicate that each enzyme has a specific glycosyl transfer mechanism on the same receptor molecule, and each enzyme synthesizes a variety of glycosyl transfer products. In addition, since the donor molecule is relatively inexpensive, there is an obvious advantage of using DGAS.

Glycosyl transfer yield of catechin β-D-fucopyranoside by cellulase from Aspergillus niger was determined to be 26%, while catechin 7-O- by α-glucosidase from Morphilus as Bacillus stea The mixture of α-D-glucopyranoside and catechin-5-O-α-D-glucopyranoside was measured at 20%. The yield of catechin glycosyl transfer by DGAS was higher than 97% based on the added catechin, which was independent of the concentration of receptor to donor ratio greater than 1: 1. The efficiency of DGAS glycosyl transition was higher than in the prior art.

Claims (5)

A method for producing a catechin derivative represented by the following formula (I), characterized by adding amylosucrase to a reaction solution containing sucrose and catechin as a substrate and enzymatically reacting at 35 ° C.
Formula 1

Wherein R is

to be. The method of claim 1,
The amylosucrase is a method for producing a catechin derivative, characterized in that amylosucrase derived from Deinococcus geothermalis ( Deinococcus geothermalis ). The method of claim 1,
The molar ratio of sucrose to catechin is 1: 1 to 10: 1. The method of claim 1,
Method for producing a catechin derivative, characterized in that the amylosucrase is used at a concentration of 50 to 500 U / mL. 5. The catechin derivative according to claim 1, wherein the catechin derivative is catechin-3′-O-α-D-glucopyranoside, catechin-3′-O-α-D-maltoside, catechin- 3′-O-α-D-maltotrioside, catechin-3′-O-α-D-malto-oligosaccharide, a method for producing a catechin derivative, characterized in that any one.

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