JP2023181589A - Method for producing monoglycoside of resveratrol - Google Patents

Abstract

To provide a novel method for producing a monoglycoside of resveratrol.SOLUTION: A method for producing a monoglycoside of resveratrol according to the present invention comprises e.g. protein consisting of an amino acid sequence with the SEQ ID NO: 1 to act on a system comprising resveratrol and saccharides. This production method can yield resveratrol 4'-O-α-glycoside represented by the formula (1), where R1 represents a sugar residue bound at the α-position.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a monoglycoside of resveratrol.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenolic compound found in wine, has been studied as having physiological functions related to the French paradox. In particular, trans-resveratrol is effective in the treatment of various diseases or conditions such as type 2 diabetes, obesity, Alzheimer's disease, osteoporosis, atherosclerosis, cardiovascular disease, and cancer, making it a useful dietary supplement. It is used as a sex ingredient. However, resveratrol is poorly soluble in water and has low stability under high pH conditions, high temperature conditions, and light conditions, so its bioavailability and industrial applications have been limited.

In order to increase the water solubility of resveratrol and improve various stability properties, it is effective to convert resveratrol into a glycoside. As a method for producing resveratrol glycosides, for example, a method in which cyclodextrin glucanotransferase (CGTase) is applied to a system containing resveratrol and maltose is known (see Non-Patent Documents 1 and 2). . According to this production method, the glycosides of resveratrol include 3-O-α-glucoside, 3-O-α-maltoside, 4'-O-α-glucoside, and 4'-O-α-maltoside. can be obtained.

Bolton, G.W. et al., Science, 232, 983-985 (1986) Torres, P. et al., Adv. Synth. Catal., 353, 1077-1086 (2011)

Generally, the physiological function of glycosides can vary depending on the position of sugar attachment. For example, resveratrol 4'-O-α-glucoside is known to have higher antioxidant activity than resveratrol 3-O-α-glucoside. However, conventionally, it has been difficult to selectively glycosylate the 4'-position of resveratrol.

Therefore, an object of the present invention is to provide a novel method for producing a monoglycoside of resveratrol.

Specific means for solving the above problems include the following embodiments.
<1> A method for producing a monoglycoside of resveratrol, which comprises allowing any of the enzyme proteins listed below (a) to (c) to act on a system containing resveratrol and saccharides.
(a) A protein consisting of the amino acid sequence of SEQ ID NO: 1.
(b) A protein consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, or added to the amino acid sequence of (a) above, and which has glycosyltransferase activity.
(c) A protein having 90% or more sequence identity with the amino acid sequence of (a) above and having glycosyltransferase activity.

［式中、Ｒ^１は、α位で結合した糖残基を示す。］ <2> The method for producing a resveratrol monoglycoside according to <1>, wherein the resveratrol monoglycoside is represented by the following formula (1).

[In the formula, R ¹ represents a sugar residue bonded at the α-position. ]

<3> The method for producing a monoglycoside of resveratrol according to <2>, wherein the sugar residue is a glucose residue.

According to the present invention, a novel method for producing a monoglycoside of resveratrol can be provided.

FIG. 3 is a diagram showing the SDS-PAGE results of His-tag fused RPG. Lane 1 shows His-tag fused RpG I purified from E. coli (Rosetta 2 (DE3)) into which plasmid (pET-21b/His-tag-RpG I) was introduced, and lane 2 shows plasmid (pET-21b/His-tag-RpG I). His-tag-RpG II) purified from E. coli (Rosetta 2 (DE3)) introduced with His-tag fusion RpG II is shown, and lane M shows a protein ladder. FIG. 3 shows HPLC chromatograms of reaction mixtures of cyclodextrin glucanotransferase (CGTase) or purified His-tag fused RPG. The upper row shows the reaction mixture of CGTase, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. P1, P2, P3, P4, and P5 are resveratrol, 3-O-α-glucoside, 3-O-α-maltoside, 4'-O-α-glucoside, and 4'-O-α-, respectively. Corresponds to maltoside. FIG. 3 is a diagram showing the influence of pH on the enzymatic activity of RpG I. Filled circles, filled triangles, and filled squares indicate sodium citrate buffer, sodium acetate buffer, and potassium phosphate buffer (KPB), respectively, and open circles, open triangles, and open squares indicate Tris-HCl buffer, heavy duty solution, respectively. Sodium carbonate buffer and sodium phosphate buffer are shown. Each value represents the mean ± standard deviation of three independent experiments. FIG. 3 is a diagram showing the pH stability of RpG I enzyme activity. Filled circles, filled triangles, and filled squares indicate sodium citrate buffer, sodium acetate buffer, and potassium phosphate buffer (KPB), respectively, and open circles, open triangles, and open squares indicate Tris-HCl buffer, heavy duty solution, respectively. Sodium carbonate buffer and sodium phosphate buffer are shown. Residual activity means the ratio (%) to the enzyme activity before treatment with KPB (pH 7.0). Each value represents the mean ± standard deviation of three independent experiments. FIG. 3 shows the effect of temperature on the enzymatic activity of RpG I. Each value represents the mean ± standard deviation of three experiments. FIG. 3 is a diagram showing the temperature stability of RpG I enzyme activity. Residual activity means the ratio (%) to the enzyme activity at 4°C before treatment. Each value is expressed as the mean ± standard deviation of three independent experiments. FIG. 3 shows the influence of Li ⁺ on the glucosylation activity of resveratrol by RpG I. Gray bars and black bars indicate 4'-O-α-glucoside and 3-O-α-glucoside, respectively. Each value represents the average of three independent experiments, and error bars represent the standard deviation of 4'-O-α-glucoside. FIG. 3 shows the influence of K ⁺ on the glucosylation activity of resveratrol by RpG I. Gray bars and black bars indicate 4'-O-α-glucoside and 3-O-α-glucoside, respectively. Each value represents the average of three independent experiments, and error bars represent the standard deviation of 4'-O-α-glucoside. FIG. 3 is a diagram showing the time course of glucosylation of resveratrol by RpG I. Glycosylation of resveratrol with RpG I was performed in 20 mmol/L Tris-HCl buffer (pH 8.0) containing 400 mM Li ⁺ at 40°C. Black circles, black triangles, and black squares indicate the concentrations of 4'-O-α-glucoside, 3-O-α-glucoside, and 4'-O-α-maltoside, respectively. Each value was calculated using the Piceido standard curve and represents the mean ± standard deviation of three independent experiments. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of caffeic acid, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of ferulic acid, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of 6-gingerol, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of daidzein, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of (S)-equol, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of genistein, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II. FIG. 3 shows HPLC chromatograms of reaction mixtures of standard substances or purified His-tag fused RPG. The upper row shows the standard substance of naringenin, the middle row shows the reaction mixture of RpG I, and the lower row shows the reaction mixture of RpG II.

Hereinafter, specific embodiments to which the present invention is applied will be described.
In this specification, a numerical range indicated using "~" indicates a range that includes the numerical values written before and after "~".

The method for producing a curcuminoid monoglycoside according to the present embodiment includes causing any of the following enzyme proteins (a) to (c) to act on a system containing curcuminoids and saccharides.
(a) A protein consisting of the amino acid sequence of SEQ ID NO: 1.
(b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) above, and which has glycosyltransferase activity.
(c) A protein having 90% or more sequence identity with the amino acid sequence of (a) above and having glycosyltransferase activity.

The protein (a) above is a protein consisting of the amino acid sequence of SEQ ID NO: 1. This protein is a protein known as α-glucosidase produced by Rhizobium pusense (GenBank accession protein ID: SDE36953). In this specification, the protein (a) above is also referred to as RpG I.

The protein (b) above is a protein that consists of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, or added to the amino acid sequence (a) above, and has glycosyltransferase activity. . The position at which the amino acid residue is substituted, deleted, or added is not particularly limited as long as the glycosyltransferase activity is maintained. Furthermore, the number of amino acid residues to be substituted, deleted, or added is not particularly limited as long as the glycosyltransferase activity is maintained. The number of amino acid residues to be substituted, deleted, or added may be, for example, 1 to 55, 1 to 45, 1 to 35, or 1 to 55. The number may be 25, 1 to 15, or 1 to 5.

When replacing any amino acid residue with another amino acid residue, it is generally preferable that the properties of the amino acid side chain are preserved before and after the substitution. When classifying amino acids by the properties of their side chains, for example, hydrophilic amino acids (D, E, K, R, H, S, T, N, Q); hydrophobic amino acids (A, G, V, I, L); , F, Y, W, M, C, P); acidic amino acids (D, E); basic amino acids (K, R, H); amino acids with aliphatic side chains (A, G, V, I, L ); Amino acids with aromatic side chains (F, Y, W); Amino acids with sulfur atom-containing side chains (M, C); etc. show).

The protein (c) above is a protein that has 90% or more sequence identity with the amino acid sequence of (a) above and has glycosyltransferase activity. The sequence identity with the amino acid sequence of (a) above may be 92% or more, 94% or more, 96% or more, or 98% or more.

Note that "sequence identity" refers to the identity between sequences when two amino acid sequences are aligned, for example, using the BLAST program (www.ncbi.nlm.nih.gov/Blast/cgi). It can be calculated by

The enzyme proteins (a) to (c) above can be produced by known methods using transformants capable of expressing the proteins.

The glycosyl donor saccharide is not particularly limited, but those having an α-1,4-glycosidic bond are preferred, and maltose is more preferred.

The conditions under which any of the enzyme proteins (a) to (c) above are allowed to act on the system containing resveratrol and sugars are not particularly limited. From the viewpoint of improving the yield of monoglycosides of resveratrol, the pH is preferably in the range of 5 to 11, and the temperature is preferably in the range of 30 to 60°C.

According to the method for producing a curcuminoid monoglycoside according to the present embodiment, resveratrol 4'-O-α-glycoside represented by the following formula (1) can be produced with high selectivity.

In the above formula (1), the sugar residue represented by R ¹ means the remainder of the sugar after removing the hemiacetal (anomeric) hydroxy group. Examples of sugar residues include glucose residues.

In addition, according to any of the enzyme proteins (a) to (c) above, not only resveratrol but also other aglycones (for example, caffeic acid, ferulic acid, 6-gingerol, naringenin, etc.) Glycosides can be produced with high regioselectivity.

Examples of the present invention will be described below, but the scope of the present invention is not limited to these Examples.

<Method>
[Cloning of α-glucosidase]
Two types of α-glucosidase genes (RpG I gene and RpG II gene) possessed by Rhizobium pusense JCM 16209 ^T strain were purchased from Genbank, and for RpG I gene (GenBank accession protein ID: SDE36953), primers of SEQ ID NO: 2 and 3 were used. The RpG II gene (GenBank accession protein ID: SDE53578) was amplified by PCR using the primers of SEQ ID NOs: 4 and 5, respectively. These primers were designed to express His-tagged α-glucosidase.
(RpG I gene)
Forward primer: 5'-GTTTAACTTTAAGAAGGAGATATACATATGACCGCTTCGGTGACTTC-3' (SEQ ID NO: 2)
Reverse primer: 5'-CGGATCTCAGTGGTGGTGGTGGTGGTGCTCGGCGTAACGGGCGAAGA-3' (SEQ ID NO: 3)
(RpG II gene)
5'-GTTTAACTTTAAGAAGGAGATATACATATGACGTTATCCCATGCCCC-3' (SEQ ID NO: 4)
5'-CGGATCTCAGTGGTGGTGGTGGTGGTGCTCTTCAATGCTCCCGCAAA-3' (SEQ ID NO: 5)

PCR was performed using PrimeSTAR GXL DNA polymerase (Takara Bio) under conditions of 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 1.7 minutes. The expression vector pET-21b for Escherichia coli was treated with restriction enzymes Nde I and Xho I, purified by gel electrophoresis, mixed with the PCR product, and used a cloning kit (In-Fusion HD Cloning Kit; Takara Bio). It was fused. Then, the obtained plasmids (pET-21b/His-tag-RpG I and pET-21b/His-tag-RpG II) were transformed into Escherichia coli (Rosetta 2 (DE3); Merck).

[Expression and purification of recombinant RPG]
Recombinant E. coli was inoculated into LB medium (500 mL) containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol at 37° C. at 95 strokes/min until the OD ₆₀₀ of the culture reached 0.6. Shaking culture was carried out under these conditions for about 3 hours. Gene expression was induced by adding isopropylthiogalactoside (IPTG) at a final concentration of 1 mmol/L. Furthermore, the recombinant E. coli was cultured at 16°C for 1 day. Gene-induced cells were collected by centrifugation, washed twice with 0.85% NaCl, and stored at 20°C until use.

Frozen cells (5 g, wet weight) were suspended in 10 mL of binding buffer and sonicated (5 minutes x 4 times). As the binding buffer, 20 mmol/L potassium phosphate buffer (KPB; pH 7.0) containing 5 mmol/L imidazole was used. Cell debris was removed by centrifugation at 14,400×g for 1 hour and 100,000×g for 90 minutes, and the supernatant was used as cell-free extract (CFE). CFE was filtered using a membrane filter with a pore size of 0.45 μm (Millex-HPF HV; Merck). The enzyme was purified from CFE using an fast protein liquid chromatography system (AKTAexplorer; Cytiva) equipped with a HisTrap™ HP (column volume 5 mL; Cytiva). The column was equilibrated with binding buffer and the adsorbed RPG was eluted using elution buffer (20 mmol/L KPB, pH 7.0) containing 500 mmol/L imidazole. Fractions containing RPG were collected, desalted, and concentrated using a Vivaspin Turbo 15 (Sartorius). Afterwards, the buffer was exchanged with 20 mmol/L KPB (pH 7.0). Finally, the solution containing RpG was mixed with an equal volume of glycerol and stored at −20° C. until use. Finally, the enzyme solution was replaced with 20 mmol/L KPB (pH 7.0) using Vivaspin Turbo 15 to remove glycerol that may function as a glycosyl acceptor. All operations were performed at 4°C.

[Enzyme assay of His-tag fused RPG]
A mixed solution (0.5 mL) consisting of 0.2 mg/mL RpG, 5 mmol/L aglycone suspended in methanol, 1 mol/L maltose, and 20 mmol/L Tris-HCl (pH 8.0) was heated under light shielding for 1200 strokes/ Incubate at 40° C. for 30 minutes with shaking. The reaction was stopped by adding 50 μL of 1N HCl. The resulting solution was centrifuged at 4° C. and 20,000×g for 5 minutes, and the supernatant was analyzed using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Unless otherwise stated, α-glucosidase activity was assessed by measuring the amount of glucosides.

[Effect of pH and temperature on glycosylation activity of RpG I]
To determine the optimal temperature and pH of purified recombinant RPG I, enzyme assays were performed using buffers (20 mmol/L) of various pH from 20 to 60 °C in 10 °C increments. did. As buffers, sodium citrate buffer (pH 2.5, 3.0, and 3.5), sodium acetate buffer (pH 3.5, 4.0, 4.5, 5.0, and 5.5) , KPB (pH 6.5, 7.0, 7.5, and 8.0), Tris-HCl buffer (pH 7.5, 8.0, 8.5, and 9.0), sodium bicarbonate buffer (pH 9.5, 10.0, 10.5, and 11.0) and sodium phosphate buffer (pH 11.5, 12.0, and 12.5).

To assess pH stability, enzyme solutions were preincubated on ice for 30 min using buffers of various pH (50 mmol/L) as described above. In addition, to evaluate thermal stability, the enzyme solution was preincubated at 4°C (on ice) or from 20°C to 80°C in 10°C increments using 20 mmol/L KPB (pH 7.0). Enzyme activity was assayed in 20 mmol/L Tris-HCl buffer (pH 9.0, except for experiments at optimum pH) for 30 minutes at 50°C (except for experiments at optimum temperature).

[Influence of monovalent cations on glycosylation activity of RpG I]
The effect of monovalent cations (10 mmol/L) on the glycosylation activity of RpG I was evaluated in 20 mmol/L Tris-HCl buffer (pH 8.0) at 40°C for 30 min. Furthermore, various concentrations of Li ⁺ (5, 10, 50, 100, 200, 400, 600, 800, and 1000 mmol/L) and K ⁺ (5, 10, 50, 100, 200, and 400 mmol/L) The impact was also evaluated.

[Influence of oligosaccharides on glycosylation activity of RpG]
from 0.2 mg/mL RpG, 5 mmol/L resveratrol suspended in methanol, 1 mol/L oligosaccharides (maltose, sucrose, trehalose, lactose, and cellobiose), and 20 mmol/L Tris-HCl (pH 8.0). The mixture (0.5 mL) was incubated at 40° C. for 30 minutes while shaking at 1200 strokes/min in the dark. The reaction was stopped by adding 50 μL of 1N HCl. The resulting solution was centrifuged at 4° C. and 20,000×g for 5 minutes, and the supernatant was analyzed using HPLC equipped with a COSMOSIL column and an SPD-M40A photodiode array detector. Furthermore, the hydrolytic activity of RpG I and RpG II on maltose in the absence of aglycone and the effect of 400 mmol/L Li ⁺ on RpG I were evaluated using HPLC equipped with an ULTRON column to determine the free glucose released. This was investigated by estimating the concentration of

[Specificity of glycosylation activity for various aglycones as glycosyl acceptors]
A mixed solution (0.5 mL) consisting of 0.2 mg/mL RpG, 5 mmol/L aglycone suspended in methanol, 1 mol/L maltose, and 20 mmol/L Tris-HCl (pH 8.0) was heated under light shielding for 1200 strokes/ Incubate at 40° C. for 30 minutes with shaking. The reaction was stopped by adding 50 μL of 1N HCl. The resulting solution was centrifuged at 4° C. and 20,000×g for 5 minutes, and the supernatant was analyzed using HPLC equipped with an SPD-M40A photodiode array detector and LC-MS. Furthermore, caffeic acid, naringenin, and resveratrol glucosides were purified from the reaction mixture, and their structures were analyzed using nuclear magnetic resonance (NMR) to identify the glucose binding positions.

[HPLC analysis]
Quantitative analysis of all aglycones and glycosides was performed using an HPLC system (Shimadzu) equipped with a COSMOSIL 5C18 PAQ column (4.6 x 150 mm; Nacalai tesque) or an ULTRON PS-80H column (8.0 x 300 mm; Shinwa Chemical Industries). It was carried out using When using a COSMOSIL column, 5.7×10 ⁻² % (v/v) acetic acid (eluent A) and acetonitrile (eluent B) were used for gradient elution. The flow rate was 1.0 mL/min. The gradient conditions are as follows. 0-5 minutes: Eluent B 10%, 5-16 minutes: Eluent B 10-80% linear gradient, 16-17 minutes: Eluent B 80-100 linear gradient, 17-22 minutes: Eluent B 100%, 22-23 min: Eluent B 100-10% linear gradient, 23-27 min: Eluent B 10%. The injector needle of the autosampler was cleaned with methanol to prevent coagulation of insoluble components after sample injection. An SPD-M10A, M20A, or M40A photodiode array detector (Shimadzu) was used to monitor the eluate. When using the ULTRON column, the mobile phase was Milli-Q water and the flow rate was 1.0 mL/min. A refractive index detector (RID-10A; Shimadzu) was used to monitor the eluate.

[LC-MS analysis]
An LC-MS system (Shimadzu) equipped with a COSMOSIL 5C18 PAQ column (4.6 x 150 mm; Nacalai tesque) was used with 5.7 x 10 ^-2 % (v/v) acetic acid (eluent A) and acetonitrile ( Gradient elution was performed using eluent B). The flow rate was 0.2 mL/min. The gradient conditions are as follows. 0-15 minutes: Eluent B 10%, 15-37 minutes: Eluent B 10-100% linear gradient, 37-57 minutes: Eluent B 100%, 57-59 minutes: Eluent B 100-10%. Linear gradient of 59-70 minutes: Eluent B 10%. The autosampler injector needle was cleaned with methanol after sample injection. An SPD-M20A photodiode array detector (Shimadzu) and LCMS-2020 (Shimadzu) were used to monitor the eluate. Detection was performed using negative mode electrospray ionization (ESI). The MS conditions are as follows. Interface temperature: 350°C, desolvation line (DL) temperature: 250°C, nebulizer gas flow rate: 1.5 L/min, heat block temperature: 200°C, drying gas flow rate: 10 L/min.

[Purification of glucosides for NMR analysis]
Resveratrol 4′-O-α-glucoside, CaUK1 (caffeic acid glucoside), CaUK2 (caffeic acid glucoside), and NUK2 (naringenin glucoside) were prepared using a COSMOSIL 5C18 PAQ column (20 × 250 mm; Nacalai tesque). Purification was performed using a HPLC system (Shimadzu). For isocratic elution, a mixture of 5.7×10 ⁻² % (v/v) acetic acid (eluent A) and acetonitrile (eluent B) was used. For resveratrol 4'-O-α-glucoside, the concentration of eluent B was 20%, for CaUK1 and CaUK2, the concentration of eluent B was 8%, and for NUK2, the concentration of eluent B was 20%. The concentration of was set to 30%. An SPD-M10A photodiode array detector (Shimadzu) was used to monitor the eluate.

[NMR analysis]
The purified product was analyzed using an NMR device (500 MHz, Avance 500; Bruker), and confirmed by ¹ H-NMR measurement and two-quantum filter ¹ H- ¹ H shift correlation two-dimensional NMR measurement (DQF-COSY). Chemical shifts were assigned relative to the solvent signal. Methanol-d was used as the solvent. The diameter of the sample tube was 5 mm.

<Results>
[Purification and reaction of recombinant His-tag fused RPG]
Recombinant E. coli expressing His-tag fused RpG I and His-tag fused RpG II were cultured, collected, disrupted, and subjected to nickel affinity chromatography to purify His-tag fused RpG I and His-tag fused RpG II. From the SDS-PAGE results, it was confirmed that both His-tag fused RpG I and His-tag fused RpG II had a molecular weight of about 60 kDa (FIG. 1A).

According to cyclodextrin glucanotransferase (CGTase) derived from microorganisms such as Thermoanaerobacter sp. and Bacillus macerans, resveratrol glycosides (3-O-α-glucoside, 3-O-α-maltoside, 4'- O-α-glucoside and 4'-O-α-maltoside) are known to be obtained (Bolton, G.W. et al., Science, 232, 983-985 (1986); Torres, P. et al. ., Adv. Synth. Catal., 353, 1077-1086 (2011)). Therefore, the CGTase reaction mixture was used as a standard substance to confirm the glucose binding position and degree of polymerization of resveratrol glucoside produced by RpG (FIG. 1B). In HPLC analysis of the CGTase reaction mixture, peaks appeared consecutively at 10.78 minutes, 11.30 minutes, 11.90 minutes, 12.16 minutes, and 13.83 minutes in the chromatogram; P5 (4'-O-α-maltoside), peak P4 (4'-O-α-glucoside), peak P3 (3-O-α-maltoside), peak P2 (3-O-α-glucoside), and It corresponded to peak P1 (resveratrol). By comparing these peaks with the peaks confirmed in reaction samples of RpG I and RpG II, it was found that RpG I has high glycosylation activity toward resveratrol. The proportions of 4'-O-α-glucoside, 3-O-α-glucoside, and 4'-O-α-maltoside produced by RpG I were 98.3%, 1.6%, and 0.9%, respectively. It was 1%. On the other hand, RpG II showed very low activity, and the 4'-position selectivity calculated from the peak area of the HPLC chromatogram was 65.6%.

[Effect of pH and temperature on glycosylation activity of RpG I]
The effect of pH and temperature on the glycosylation activity of RpG I towards resveratrol using maltose as the glycosyl donor was investigated from pH 2.5 to pH 12.5 and from 20°C to 60°C. Maximum activity to produce 4'-O-α-glucoside was observed at pH 8.0 (using KPB) and 50°C (Figure 2A, Figure 2C). Glycosylation activity was stable up to 40°C, but rapidly lost at temperatures above 50°C (Fig. 2D). In pH stability studies, the enzyme maintained maximum activity between pH 3.5 and pH 11.0 (Figure 2B). Regarding regioselectivity, no difference in reaction temperature was observed, and 4'-position selectivity was maintained at 98.6% or higher. However, as the reaction pH decreased, the 4'-position selectivity decreased and finally reached 91.1% at pH 3.5 (using sodium citrate buffer). Conversely, as the reaction pH increased, the 4'-position selectivity also increased and finally reached 99.7% at pH 9.5, although some by-products were detected. This is presumed to be because resveratrol is unstable under alkaline conditions (Zupancic, S. et al., Eur. J. Pharm. Biopharm., 93, 196-204 (2015)).

[Influence of monovalent cations on glycosylation activity of RpG I]
According to the sequence-based classification of carbohydrate hydrolases (GH), bacterial α-glucosidases are classified into GH family 13 (GH13). Previous studies have shown that the α-glucosidase activity of Halomonas sp. H11 strain and Ensifer adhaerens NBRC 100388 ^T strain is enhanced by NH ₄ ⁺ and K ⁺ (Ojima, T. et al., Appl. Environ Microbiol., 78, 1836-1845 (2012); Suzuki, T. et al., Biocatal. Agric. Biotechnol., 30, 101837 (2012)). Therefore, the influence of various monovalent cations (10 mM) on the activity of 0.2 mg/mL RpG I was also evaluated. CsCl, LiCl, RbCl, NH ₄ Cl, NaCl, and KCl were used as monovalent cation salts. When 20 mmol/L KPB (pH 8.0) was used as the optimal buffer (see Figure 2A), the activity was slightly inhibited by all cations (Table 1). However, when 20 mmol/L Tris-HCl buffer (pH 8.0) was used, RpG I activity was enhanced to 135% by Cs ⁺ and Li ⁺ (Table 2).

Furthermore, with various concentrations of Li ⁺ (5, 10, 50, 100, 200, 400, 600, 800, and 1000 mmol/L) and K ⁺ (5, 10, 50, 100, 200, and 400 mmol/L) The effects were evaluated (Figure 3A, Figure 3B). LiCl and KCl were used as Li salt and K salt. The amount of 4'-O-α-glucoside increased in a concentration-dependent manner up to a 2.5-fold increase up to a Li ⁺ concentration of 400 mmol/L, and decreased at higher concentrations. In contrast, the amount of 3-O-α-glucoside increased in a concentration-dependent manner up to a Li ⁺ concentration of 1 mol/L. Activity was slightly suppressed with K ⁺ . The 4′-position selectivity increased concentration-dependently up to a Li ⁺ concentration of 100 mmol/L, reaching a maximum of 99.1%, and decreased to a minimum of 96.0% at higher concentrations.

Note that the effect of Li ⁺ differed depending on the type of RpG I, RpG II, and aglycon. This indicates that effective cations for GH13 vary depending on the combination of enzyme structure, glycosyl donor, glycosyl acceptor, and reaction conditions.

[Influence of oligosaccharides on glycosylation activity of RpG I]
The glycosylation activity of RpG I was evaluated using various oligosaccharides by measuring the concentration of resveratrol 4'-O-α-glucoside (Table 3). The highest activity was obtained when maltose was used as the glycosyl donor, followed by sucrose. The activity was relatively low when using trehalose, and no resveratrol 4'-O-α-glucoside was detected when using lactose and cellobiose. When maltose, sucrose, and trehalose were used, the 4' selectivity was 98.3%, 92.7%, and 94.6%, respectively, indicating that the regioselectivity of glycosylation differs depending on the type of oligosaccharide. It shows.

Furthermore, the hydrolysis activities of RpG I and RpG II towards maltose in the absence of aglycone were compared by measuring the concentration of free glucose released using HPLC equipped with an ULTRON column. As a result, the activity of RpG I was about 8 times and about 4 times higher than that of RpG II and the activity of RpG I when Li ⁺ was included, respectively (Table 4). This suggested that Li ⁺ enhances the glycosylation activity of RpG I by suppressing the hydrolytic activity in addition to activating the enzyme.

[Enzymatic synthesis of resveratrol glucoside using RpG I under optimal conditions]
The time course of glycosylation of 5 mmol/L resveratrol was measured using 0.2 mg/mL RpG I in 20 mmol/L Tris-HCl buffer at 40 °C containing 400 mM Li ⁺ (Figure 4 ). The maximum molar yield of resveratrol 4′-O-α-glucoside reached 41.6% at 30 minutes, and the product concentration was 2.08 mmol/L. From this point onwards, hydrolysis reactions became dominant. In contrast, 3-O-α-glucoside and 4′-O-α-maltoside were synthesized in a time-dependent manner, with a corresponding decrease in the proportion of 4′-O-α-glucoside.

[Substrate specificity of RpG for various aglycones]
To assess the substrate specificity of RpG, reaction mixtures containing each polyphenol compound were prepared and analyzed by HPLC to identify the products (Figures 5A to 5G). Since an unknown peak (UK) with absorption spectra similar to each aglycon was detected in the reaction mixture, RpG I was determined to contain all the aglycones evaluated (resveratrol, caffeic acid, ferulic acid, 6-gingerol, It appeared to have glycosylation activity on daidzein, (S)-equol, genistein, and naringenin). On the other hand, RpG II showed almost no activity against any of the aglycones evaluated, making it impossible to accurately evaluate regioselectivity.

[LC-MS analysis of RpG I reaction mixture]
The RpG I reaction mixture was analyzed by LC-MS and confirmed to be glucosides with reference to MS spectra (not shown). As a result, CaUK1 (caffeic acid glucoside), CaUK2 (caffeic acid glucoside), FUK1 (ferulic acid glucoside), 6GUK2 (6-gingerol glucoside), DUK1 (daidzein glucoside), DUK2 (daidzein glucoside), EUK2 ((S )-equol glucoside), GUK1 (genistein glucoside), GUK2 (genistein glucoside), NUK1 (naringenin glucoside), and NUK2 (naringenin glucoside) are monoglucosides formed by the transfer of a single glucose molecule to each aglycone. It turned out to be. In contrast, 6GUK1 (6-gingerol glucoside) and EUK1 ((S)-equol glucoside) corresponded to diglucosides.

[Determination and estimation of glucose binding position using α-glucoside or β-glucoside standard]
LC-MS analysis revealed that the regioselectivity of RpG I for 6-gingerol and (S)-equol was 100%. The present inventors have reported that 5-O-α-glucosylgingerol is produced from 6-gingerol by a microbial-derived glycosyltransferase (Matsumoto, R. et al., J. Mol. Catal. B Enzym., 133, S200-S203 (2016)). Therefore, the glucose binding position of 6GUK2 was determined using purified 5-O-α-glucosylgingerol as a standard substance. The results showed that 6GUK2 was 5-O-α-glucosyl gingerol and that RpG I glycosylated the secondary alcohol of gingerol with high regioselectivity (see Figure 5C).

Although the anomeric isomers α-glucoside and β-glucoside have similar polarity and similar retention times on HPLC, α-glucoside elutes slightly earlier than β-glucoside. Therefore, using daidzein 7-O-β-glucoside (Daidzin), genistein 4'-O-β-glucoside (Sophoricoside), and genistein 7-O-β-glucoside (Genistin) as standard substances, daidzein and genistein We estimated the glucose binding position of . As a result, the retention times of DUK1 and daidzin were the same. Therefore, DUK1 and DUK2 were estimated to be 7-O-α-glucoside and 4'-O-α-glucoside, respectively (see Figure 5D). Furthermore, the retention times of GUK1 and GUK2 were consistent with those of genistin and sophorocoside, respectively. Therefore, GUK1 and GUK2 were estimated to be 7-O-α-glucoside and 4'-O-α-glucoside, respectively (see Figure 5F).

[Structural analysis of glucoside using NMR]
To gain further insight into the regioselectivity of RpG I, resveratrol 4'-O-α-glucoside, CaUK1, CaUK2, and NUK2, were purified from the reaction mixture and subjected to NMR analysis. According to ¹ H-NMR data of aglycones and glucosides, the chemical shift values around the glycosylation site shift toward the downfield side. By referring to this shift, the glucose binding position was identified. As a result, the chemical structure of resveratrol 4'-O-α-glucoside was confirmed. Additionally, CaUK1, CaUK2, and NUK2 were identified as caffeic acid 4-O-α-glucoside, caffeic acid 3-O-α-glucoside, and naringenin 4'-O-α-glucoside, respectively. ¹ H-NMR data of resveratrol 4'-O-α-glucoside, CaUK1, CaUK2, and NUK2 are shown below.

Resveratrol 4'-O-α-glucoside
¹ H NMR d _H (CD ₃ OD): 7.46 (2H, d, J=8.7 Hz, Hf, Hi), 7.16 (2H, d, J=8.8 Hz, Hg, Hh), 7.00 (1H, d, J =16,3 Hz, Hd or He), 6.88 (1H, d, J=16,3 Hz, Hd or He), 6.46 (2H, s, Ha, Hc), 6.17 (1H, s, Hb), 5.50 (1H, d, J=3.7 Hz, Hj), 3.86 (1H, dd, J=9.3, 9.4 Hz, Hn), 3.73 (2H, d, J=14.1 Hz, Hl), 3.65-3.68 (1H, m , Hk), 3.58 (1H, dd, J=9.7, 3.6 Hz, Ho), 3.44 (1H, dd, J=9.3, 9.6 Hz, Hm).

CaUK1: Caffeic acid 4-O-α-glucoside
¹ H NMR d _H (CD ₃ OD): 7.52 (1H, d, J=15.9 Hz, He), 7.29 (1H, d, J=8.35 Hz, Hb), 7.10 (1H, s, Ha), 7.02 ( 1H, d, J=8.0 Hz, Hc), 6.33 (1H, d, J=15.9 Hz, Hd), 5.42 (1H, d, J=3.7 Hz, Hf), 3.88 (1H, dd, J=9.4, 9.6 Hz, Hj), 3.75 (2H, d, J=23.3 Hz, Hh), 3.67-3.70 (1H, m, Hg), 3.60 (1H, dd, J=9.8, 3.8 Hz, Hk), 3.43 (1H , dd, J=9.3, 9.1 Hz, Hi).

CaUK2: Caffeic acid 3-O-α-glucoside
¹ H NMR d _H (CD ₃ OD): 7.56 (1H, d, J=15.9 Hz, He), 7.55 (1H, s, Ha), 7.18 (1H, d, J=8.4 Hz, Hc), 6.87 ( 1H, d, J=8.4 Hz, Hb), 6.32 (1H, d, J=16.0 Hz, Hd), 5.36 (1H, d, J=7.3 Hz, Hf), 3.87 (1H, dd, J=9.4, 9.6 Hz, Hj), 3.78 (2H, d, J=16.5 Hz, Hh), 3.72-3.77 (1H, m. Hg), 3.61 (1H, dd, J=9.5, 5.8 Hz, Hk), 3.42 (1H , dd, J=9.4, 9.2 Hz, Hi).

NUK2: Naringenin 4'-O-α-glucoside
¹ H NMR d _H (CD ₃ OD): 7.44 (2H, d, J=8.7 Hz, He, Hh), 7.22 (2H, d, J=8.7 Hz, Hf, Hg), 5.91 (1H, s, Ha ), 5.89 (1H, s, Hb), 5.52 (1H, d, J=3.6 Hz, Hi), 5.42 (1H, d, J=12.6, 3.1 Hz, Hf-1), 3.86 (1H, dd, J =9.3, 9.3 Hz, Hm), 3.72 (2H, d, J=15.7 Hz, Hk), 3.64-3.66 (1H, m, Hj), 3.58 (1H, dd, J=9.7, 3.5 Hz, Hn), 3.43 (1H, dd, J=9.3, 9.3 Hz, Hl), 3.13 (1H, d, J=12.7 Hz, Hg-1), 3.09 (1H, d, J=12.7 Hz, Hg-2), 2.75 ( 1H, d, J=17.1, 3.2 Hz, Hf-2).

[Regioselectivity of RpG I for polyphenol compounds]
Finally, for each monoglucoside except resveratrol glucoside, the regioselectivity was calculated from the peak area of the MS chromatogram (Tables 5 and 6). Since the regioselectivity of daidzein, (S)-equol, and genistein monoglycerides has not been confirmed, Table 6 shows the proportions of major monoglycerides. In this experiment, α-glucosidase (RpG I) derived from Rhizobium pusense JCM 16209 ^T strain glycosylated resveratrol with high 4' position selectivity (98.3%), resulting in resveratrol 4'-O-α- It was revealed that glucosides are produced. The 4' position of resveratrol corresponds to the para position of the phenyl group. As a result of studies on various polyphenol compounds, it became clear that para-selective glycosylation of resveratrol can be generalized to many polyphenol compounds. In fact, both caffeic acid and naringenin were glycosylated with high para-selectivity.

Claims (3)

A method for producing a monoglycoside of resveratrol, the method comprising allowing any of the enzyme proteins listed below (a) to (c) to act on a system containing resveratrol and saccharides.
(a) A protein consisting of the amino acid sequence of SEQ ID NO: 1.
(b) A protein consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, or added to the amino acid sequence of (a) above, and which has glycosyltransferase activity.
(c) A protein having 90% or more sequence identity with the amino acid sequence of (a) above and having glycosyltransferase activity. The method for producing a resveratrol monoglycoside according to claim 1, wherein the resveratrol monoglycoside is represented by the following formula (1).

[In the formula, R ¹ represents a sugar residue bonded at the α-position. ] The method for producing a monoglycoside of resveratrol according to claim 2, wherein the sugar residue is a glucose residue.

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