Method for producing alcohol glycoside

Abstract

This method for producing an alcohol glycoside includes bringing a glycosyltransferase, an aromatic alcohol represented by general formula (1), an ascorbic acid derivative, and a monosaccharide donor into contact with each other to form an alcohol glycoside. In general formula (1), R 1 represents a hydroxy group, and n represents an integer of 1-5.

Description

Method for producing alcohol glycosides

[0001] The present disclosure relates to a method for producing an alcohol glycoside.

[0002] Glycosyltransferases are enzymes that catalyze the transfer of sugar residues from sugar donors, such as sugar nucleotides, to sugar acceptors to form glycosidic bonds. They are used in industrial enzymatic glycoside production processes.

[0003] Among many glycosides, alcohol glycosides are useful compounds that are widely used as antioxidants, antimicrobial agents, anti-inflammatory agents, foods, cosmetics, etc. In particular, alcohol glycosides such as arbutin, which are produced from aromatic alcohols (e.g., hydroquinone) and monosaccharide donors, are expected to be produced efficiently due to their usefulness.

[0004] Alcohol glycosides are formed by the binding of a non-sugar alcohol moiety to a sugar moiety through an enzymatic reaction using glycosyltransferase. For example, Patent Document 1 describes a method for producing hydroquinone glycosides from hydroquinone and UDP-glucose using a mutant glycosyltransferase (UGT72B1). Patent Document 2 describes a method for producing hydroxytyrosol glycoside (hydroxytyroxaside) from hydroxytyrosol and UDP-glucose through a regioselective enzymatic reaction using mutant glycosyltransferases (Oft8GT1 and Oft8GT3). Patent Document 3 describes that a method for producing an O-linked glucuronide conjugate from 2-aminophenol and UDP-glucuronic acid can be used to evaluate the activity of mutant glycosyltransferases. Patent Document 4 describes a method for producing flavonoid glycosides in the presence of cyclodextrin, a flavonoid inclusion compound, using a glycosyltransferase such as a glycosyltransferase. Non-Patent Document 1 describes a method for producing hydroquinone glycosides from hydroquinone and UDP-glucose using a mutant glycosyltransferase (UGT72B11).

[0005] Patent Publication No. 2007-525966 Chinese Patent No. 114317480 US Patent Application Publication No. 2006 / 0019326 International Publication No. 2019 / 021510

[0006] Simone Witte, SoWa Moco, Jacques Vervoort, Ulrich Matern, Stefan Martens, Planta (2009) 229:1135-1146.

[0007] However, in conventional methods for producing alcohol glycosides, the activity of producing alcohol glycosides tends to decrease under conditions of high substrate concentrations (e.g., substrate concentrations of 50 mmol / L or higher). For example, Non-Patent Document 1 describes that a mutant glycosyltransferase (UGT72B11) has low activity in producing hydroquinone glycosides from hydroquinone and UDP-glucose, and that hydroquinone is unsuitable as a substrate.

[0008] In view of the above circumstances, the present disclosure relates to providing a method for producing an alcohol glycoside that has excellent alcohol glycoside activity even under conditions of high substrate concentration (for example, a substrate concentration of 50 mmol / L or more).

[0009] The present disclosure includes the following aspects: <1> A method for producing an alcohol glycoside, comprising contacting a glycosyltransferase with an aromatic alcohol represented by the following general formula (1), an ascorbic acid derivative, and a monosaccharide donor to form the alcohol glycoside: In general formula (1), R １represents a hydroxy group, and n represents an integer of 1 or more and 5 or less. <2> A method for producing an alcohol glycoside according to <1>, wherein the aromatic alcohol represented by general formula (1) has a molar concentration of 50 mmol / L or more. <3> A method for producing an alcohol glycoside according to <1> or <2>, wherein the molar ratio of the aromatic alcohol represented by general formula (1) to the ascorbic acid derivative (aromatic alcohol / ascorbic acid derivative) is 5 or more and 200 or less. <4> A method for producing an alcohol glycoside according to any one of <1> to <3>, wherein the aromatic alcohol represented by general formula (1) includes at least one selected from the group consisting of hydroquinone, catechol, and resorcinol. <5> A method for producing an alcohol glycoside according to any one of <1> to <4>, wherein the monosaccharide donor includes a uridine diphosphate sugar. <6> The method for producing an alcohol glycoside according to any one of <1> to <5>, wherein the glycosyltransferase has an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (UGT72B11), the amino acid sequence represented by SEQ ID NO: 2 (UGT72B1), or the amino acid sequence represented by SEQ ID NO: 3 (UGT73A10). <7> The method for producing an alcohol glycoside according to <6>, wherein the glycosyltransferase has an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (UGT72B11), the amino acid sequence represented by SEQ ID NO: 2 (UGT72B1), or the amino acid sequence represented by SEQ ID NO: 3 (UGT73A10).

[0010] According to the present disclosure, it is possible to provide a method for producing an alcohol glycoside that has excellent alcohol glycoside activity even under conditions of high substrate concentration (for example, a substrate concentration of 50 mmol / L or more).

[0011]

[0023] The following describes embodiments of the present disclosure. These descriptions and examples are intended to illustrate the embodiments and are not intended to limit the scope of the embodiments.

[0012] In the numerical ranges described in stages in this disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples.

[0013] In the present disclosure, each component may contain multiple types of corresponding substances.

[0014] In the present disclosure, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0015] In the present disclosure, the glycosyltransferase activity that obtains an alcohol glycoside from an aromatic alcohol represented by the general formula (1) described below and a monosaccharide donor is also referred to as "alcohol glycosidation activity."

[0016] In the present disclosure, a description of a nucleotide sequence may be given with a focus on the sequence on one strand even when the nucleic acid strand carrying the nucleotide sequence forms a double strand; however, in the other strand of the double strand, such a description of the sequence should be read and applied to its complementary sequence.

[0017] In the present disclosure, amino acid residues may be represented by a single alphabetic letter, for example, a tryptophan residue may be represented by "W."

[0018] <Method for Producing Alcohol Glycoside> The method for producing an alcohol glycoside of the present disclosure includes contacting a glycosyltransferase with an aromatic alcohol represented by the following general formula (1), an ascorbic acid derivative, and a monosaccharide donor to form an alcohol glycoside. In general formula (1), R １ represents a hydroxy group, and n represents an integer of 1 or more and 5 or less.

[0019] Aromatic alcohols represented by general formula (1) are more susceptible to oxidation in transglycosylation reactions than other aromatic alcohols. Furthermore, due to their molecular structure, ketone bodies formed by oxidation of aromatic alcohols represented by general formula (1) tend to preferentially act on glycosyltransferases in transglycosylation reactions. As a result, under high substrate concentrations, glycosyltransferase function as a glycosyltransferase tends to decrease, resulting in reduced alcohol glycosidation activity. On the other hand, according to the present disclosure, excellent alcohol glycoside activity is achieved even under high substrate concentrations (e.g., substrate concentrations of 50 mmol / L or higher). The mechanism of this action is not entirely clear, but is presumed to be as follows. In the production method of the present disclosure, an ascorbic acid derivative is included in the transglycosylation reaction between an aromatic alcohol represented by general formula (1) and a monosaccharide donor using a glycosyltransferase. The ascorbic acid derivative acts as an antioxidant in the reaction system, suppressing the oxidation of the aromatic alcohol represented by general formula (1). Furthermore, due to their molecular structure, ascorbic acid derivatives are likely to capture ketone bodies formed by oxidation of aromatic alcohols represented by general formula (1) in the reaction system and inhibit the action of ketone bodies on glycosyltransferases, which is thought to result in excellent alcohol glycoside activity even under conditions of high substrate concentrations.

[0020] The method for contacting the glycosyltransferase with the aromatic alcohol represented by general formula (1), the ascorbic acid derivative, and the monosaccharide donor is not particularly limited. For example, the aromatic alcohol represented by general formula (1), the ascorbic acid derivative, and the monosaccharide donor may be mixed with a culture medium containing a transformant or cell line that produces glycosyltransferase, a treated cell product, or a purified enzyme product. In this manner, the glycosyltransferase comes into contact with the aromatic alcohol represented by general formula (1) and the monosaccharide donor in the presence of the ascorbic acid derivative, and catalyzes the reaction of forming an alcohol glycoside from the aromatic alcohol represented by general formula (1) and the monosaccharide donor.

[0021] The treated cell product refers to, for example, an extract or disrupted product from a transformant, or a substrate-immobilized product thereof. Examples of the substrate for the substrate-immobilized product include resin beads. The temperature during the contact and reaction is, for example, preferably 20°C to 60°C, and more preferably 30°C to 50°C.

[0022] In one embodiment, a purified product of glycosyltransferase extracted and purified from a culture medium in which a transformant or a cell line is cultured may be mixed with a solution containing at least an ascorbic acid derivative, an aromatic alcohol represented by general formula (1), and a monosaccharide donor, and the two may be brought into contact with each other and reacted to form an alcohol glycoside.

[0023] The pH of the aqueous solution during the contact and reaction may be, for example, 6.0 or more and 8.5 or less at 23°C. The pH of the aqueous solution during the contact and reaction is, for example, preferably 6.0 or more and less than 8.0 at 23°C, more preferably 6.2 or more and 7.8 or less, and even more preferably 6.5 or more and 7.5 or less. A pH of 6.0 or more suppresses the collapse of glycosyltransferase folding, resulting in superior alcohol glycosidation activity. A pH of less than 8.0 further suppresses the oxidation of the aromatic alcohol represented by general formula (1), and further enhances the auxiliary effect of the ascorbic acid derivative, resulting in superior alcohol glycosidation activity.

[0024] The molar ratio of the aromatic alcohol represented by general formula (1) to the ascorbic acid derivative (aromatic alcohol represented by general formula (1) / ascorbic acid derivative) is preferably 1 or more and 100 or less, more preferably 5 or more and 20 or less. When the molar ratio (aromatic alcohol represented by general formula (1) / ascorbic acid derivative) is 1 or more, the ascorbic acid derivative acts more efficiently as an antioxidant in the reaction system, thereby more effectively suppressing the oxidation of the aromatic alcohol represented by general formula (1). Furthermore, the ascorbic acid derivative more efficiently captures ketone bodies formed by the oxidation of the aromatic alcohol represented by general formula (1) in the reaction system, more effectively inhibiting the action of ketone bodies on glycosyltransferase. As a result, the alcohol glycoside activity is more excellent. When the molar ratio (aromatic alcohol represented by general formula (1) / ascorbic acid derivative) is 100 or less, the action of excess ascorbic acid derivative on glycosyltransferase, which would inhibit the transglycosylation reaction, is suppressed, resulting in more excellent alcohol glycoside activity.

[0025] [Glycosyltransferase] The glycosyltransferase is not particularly limited, and known glycosyltransferases that catalyze the glycosylation of alcohols can be used. Examples of glycosyltransferases include uridine diphosphate glucosyltransferase (UGT). As UGTs, from the viewpoint of superior alcohol glycosylation activity, preferred are UGT72 family (e.g., UGT72B11, UGT72B1, UGT72E2, UGT72E1, etc.), UGT73 family (e.g., UGT73B11, UGT73A10, etc.), and mutant glycosyltransferases having one or more mutations in these UGTs. One type of glycosyltransferase may be used alone, or two or more types may be used in combination.

[0026] Alignment of glycosyltransferase sequences can be performed by pairwise alignment using the sequence homology search program BLAST (trademark), with default parameters. Sequence identity is calculated over the full length of the glycosyltransferase.

[0027] From the viewpoint of achieving superior alcohol glycosylation activity, the glycosyltransferase preferably has a sequence identity of 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 100% to the amino acid sequence represented by SEQ ID NO: 1 (UGT72B11), the amino acid sequence represented by SEQ ID NO: 2 (UGT72B1), or the amino acid sequence represented by SEQ ID NO: 3 (UGT73A10).

[0028] The method for producing glycosyltransferase is not particularly limited, and production may be performed under preferred conditions depending on the glycosyltransferase used. Examples of methods for producing glycosyltransferase include preparing a vector containing a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the glycosyltransferase, and using this vector to produce a mutant glycosyltransferase.

[0029] [Aromatic Alcohol Represented by General Formula (1)] The aromatic alcohol used in the method for producing an alcohol glycoside of the present disclosure is represented by the following general formula (1). In general formula (1), R １ represents a hydroxy group; n represents an integer of 1 or more and 5 or less, preferably an integer of 1 or more and 3 or less, more preferably 1 or 2, and even more preferably 1;

[0030] Exemplary compounds of aromatic alcohols represented by general formula (1) are shown below, but the aromatic alcohols represented by general formula (1) used in the present disclosure are not limited to these.

[0031]

[0032] The aromatic alcohol represented by general formula (1) preferably contains at least one selected from the group consisting of hydroquinone (exemplary compound 1-1), resorcinol (exemplary compound 1-2), and catechol (exemplary compound 1-3), and more preferably contains hydroquinone (exemplary compound 1-1). When the aromatic alcohol represented by general formula (1) contains at least one selected from the above group, it is thought that, compared to aromatic alcohols having three or more hydroxy groups (e.g., exemplary compounds 1-4 to 1-9), it is easier to enter the enzyme pocket of glycosyltransferase in terms of its molecular structure, and the reaction efficiency between glycosyltransferase and a monosaccharide donor is superior.

[0033] In the method for producing an alcohol glycoside of the present disclosure, a glycosyltransferase, an aromatic alcohol represented by general formula (1), an ascorbic acid derivative, and a monosaccharide donor are contacted. In this method, even if the substrate concentration (i.e., the concentration of the aromatic alcohol represented by general formula (1)) during alcohol glycoside formation is high, the alcohol glycoside activity is believed to be excellent. More specifically, the molar concentration of the aromatic alcohol represented by general formula (1) before the reaction may be 50 mmol / L or more and 400 mmol / L or less, or 100 mmol / L or more and 350 mmol / L or less.

[0034] [Ascorbic Acid Derivatives] The term "ascorbic acid derivatives" encompasses ascorbic acid and its stereoisomer, erythorbic acid, as well as alkali metal salts thereof (e.g., sodium salts and potassium salts). Examples of ascorbic acid derivatives include, but are not limited to, ascorbic acid, sodium ascorbyl phosphate, ascorbyl glucoside, magnesium ascorbyl phosphate, ascorbyl aminopropyl phosphate, disodium ascorbyl sulfate, ascorbic acid 2-phosphate, and salts thereof. One ascorbic acid derivative may be used alone, or two or more may be used in combination. Among the above, from the viewpoints of achieving superior antioxidant properties against aromatic alcohols represented by general formula (1) and facilitating capture of ketone bodies from aromatic alcohols represented by general formula (1), the ascorbic acid derivative preferably contains at least one of ascorbic acid and sodium ascorbyl phosphate, and more preferably contains ascorbic acid.

[0035] The molar concentration of the ascorbic acid derivative in the reaction system may be 1 mmol / L or more, 3 mmol / L to 100 mmol / L or less, or 5 mmol / L to 50 mmol / L or less. When the molar concentration of the ascorbic acid derivative is 1 mmol / L or more, the ascorbic acid derivative acts more efficiently as an antioxidant in the reaction system, thereby more effectively suppressing the oxidation of the aromatic alcohol represented by general formula (1). Furthermore, the ascorbic acid derivative more efficiently captures the ketone body formed by the oxidation of the aromatic alcohol represented by general formula (1) in the reaction system, more effectively inhibiting the ketone body from acting on the enzyme pocket of glycosyltransferase. As a result, the alcohol glycoside activity is more excellent. When the molar concentration of the ascorbic acid derivative is 100 mmol / L or less, the inhibition of the glycosyltransferase reaction due to the action of excess ascorbic acid derivative on glycosyltransferase is suppressed, resulting in more excellent alcohol glycoside activity.

[0036] [Monosaccharide Donor] The monosaccharide donor is not particularly limited as long as it can act as a monosaccharide donor in the glycosyltransferase-catalyzed glycosyltransferase-catalyzed glycosyltransferase reaction, and examples thereof include monosaccharide sugar nucleotides such as glucose, fructose, and galactose. Examples of sugar nucleotides include uridine diphosphate sugars. One type of monosaccharide donor may be used alone, or two or more types may be used in combination.

[0037] The monosaccharide donor preferably contains a uridine diphosphate sugar, from the viewpoint of facilitating the entry of the monosaccharide donor into the enzyme pocket and improving the reaction efficiency between the enzyme and the monosaccharide donor. Examples of the uridine diphosphate sugar include uridine diphosphate glucose, uridine diphosphate fructose, and uridine diphosphate galactose. Among these, uridine diphosphate glucose is preferred, from the viewpoint of facilitating the entry of the monosaccharide donor into the enzyme pocket and further improving the reaction efficiency between the enzyme and the monosaccharide donor.

[0038] The following examples will explain the embodiments in more detail, but the embodiments of the present disclosure are not limited to the following examples in any way.

[0039] <Obtaining glycosyltransferase> - Obtaining glycosyltransferase 1 (UGT72B11) A nucleic acid encoding the polypeptide of SEQ ID NO: 1 was codon-optimized and synthesized as a nucleic acid gene. Competent cells of E. coli strain BL21 (DE3) were transformed using the pETDuet-1 vector system into which this synthetic gene was cloned, and the cells were plated on LB agar medium containing 100 μg / mL ampicillin and cultured overnight at 37°C. The grown colonies were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured overnight with shaking at 37°C and 200 rpm. A portion of the culture was inoculated into 0.3 L of LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 200 rpm. After the optical density at 600 nm (OD600) reached 0.6-0.8, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and the cells were cultured at 30°C and 200 rpm for 18 hours with shaking. The supernatant was discarded after centrifugation (4°C, 3000G, 20 min). The obtained cell pellet was suspended in 30 mL of lysis buffer (20 mM HEPES, 300 mM sodium chloride solution (pH 7.5)) and processed on ice using a homogenizer (BRANSON SONIFER). The processed solution was centrifuged (4°C, 5000G, 30 min), and the cell debris was discarded. The obtained supernatant was purified by His tag, ultrafiltered, and substituted with 50 mM HEPES buffer (pH 7.5) to obtain a purified enzyme solution, yielding a composition containing glycosyltransferase. The sequences of the above nucleic acid genes are shown below.

[0040]

[0041] Obtaining glycosyltransferase 2 (UGT72B1) A nucleic acid encoding the polypeptide of SEQ ID NO: 2 was codon-optimized and synthesized as a nucleic acid gene. Other specifications were the same as for glycosyltransferase 1, and a composition containing glycosyltransferase 2 was obtained. The sequence of the nucleic acid gene is shown below.

[0042]

[0043] Obtaining glycosyltransferase 3 (UGT73A10) A nucleic acid encoding the polypeptide of SEQ ID NO: 3 was codon-optimized and synthesized as a nucleic acid gene. Other specifications were the same as for glycosyltransferase 1, and a composition containing glycosyltransferase 3 was obtained. The sequence of the nucleic acid gene is shown below.

[0044]

[0045] Obtaining glycosyltransferase 4 (UGT72E2) A nucleic acid encoding the polypeptide of SEQ ID NO: 4 was codon-optimized and synthesized as a nucleic acid gene. Other specifications were the same as for glycosyltransferase 1, and a composition containing glycosyltransferase 4 was obtained. The sequence of the nucleic acid gene is shown below.

[0046]

[0047] The amino acid sequences of each glycosyltransferase are shown below.

[0048] SEQ ID NO: 1 (UGT72B11): MEGTPHIAIVPSPGMGHLIPLVEFAKRLNTNHNISAIFIIPNDGPLSKSQIAFLDSLPDGLSYLILPPVNFDDLPKDTLMETRISLMVTRSVPSLRQVFKSLVAEKHMVALFIDLFGTDAFDVAIEFGVSPYVFFPSTAMVLSMFLNLPRLDQEVSCEYRDLPEPVQIPGCIPVRGEDLLDPVQDRKNDAYKWVLHNAKRYRMAEGIAVNSFQELEGGALKVLLEEEPGKPRV YPVGPLIQSGSSSDLDGSDCLRWLDSQPCGSVLYISFGSGGTLSSTQLNELAMGLELSEQRFLWVVRSPNDQPNATYFDSHGHNDPLGFLPKGFLERTKNTGFVVPSWAPQAQILS HSSTGGFLTHCGWNSILETVVHGVPVIAWPLYAEQKMNAVSLTEGLKVALRPKVGDNGIVGRLEIARVVKGLLEGEEGKGIRSRIRDLKDAAANVLGKDGCSTKTLDQLASKLKNKI

[0049] SEQ ID NO: 2 (UGT72B1): MEESKTPHVAIIPSPGMGHLIPLVEFAKRLVHLHGLTVTFVIAGEGPPSKAQRTVLDSLPSSISSVFLPPVDLTDLSSSTRIESRISLTVTRSNPELRKVFDSFVEGGRLPTALVVDLFGTDAFDVAVEFHVPPYIFYPTTANVLSFFLHLPKLDETVSCEFRELTEPLMLPGCVPVAGKDFLDPAQDRKDDAYKWLLHNTKRYKEAEGILVNTFFELEPNAIKALQEPGLDKPPVYPVG PLVNIGKQEAKQTEESECLKWLDNQPLGSVLYVSFGSGGTLTCEQLNELALGLADSEQRFLWVIRSPSGIANSSYFDSHSQTDPLTFLPPGFLERTKKRGFVIPFWAPQAQVLAHPSTGG FLTHCGWNSTLESVVSGIPLIAWPLYAEQKMNAVLLSEDIRAALRPRAGDDGLVRREEVARVVKGLMEGEEGKGVRNKMKELKEAACRVLKDDGTSTKALSLVALKWKAHKKELEQNGNH

[0050] SEQ ID NO: 3 (UGT73A10): MAETPVVTPHIAILPSPGMGHLIPLVEFSKRLIQNHHFSVTLILPTDGPVSNAQKIYLNSLPCSMDYHLLPPVNFDDLPLDTKMETRISLTVTRSLPSLREVFKTLVETKKTVALVVDLFGTDAFDVANDFKVSPYIFYPSTAMALSLFLYLPKLDETVSCEYTDLPDPVQIPGCIPIHGKDLLDPVQDRKNEAYKWVLHHSKRYRMAEGIVANSFKELEGGAIKALQEEEPGKPPVY PVGPLIQMDSGSGSKADRSECLTWLDEQPRGSVLYISFGSGGTLSHEQMIELASGLEMSEQRFLWVIRTPNDKMASATYFNVQDSTNPLDFLPKGFLEKTKGLGLVVPNWAPQAQILGH GSTSGFLTHCGWNSTLESVVHGVPFIAWPLYAEQKMNAVMLSEDIKVALRPKANENGIVGRLEIAKVVKGLMEGEEGKVVRSRMRDLKDAAAKVLSEDGSSTKALAELATKLKKKVSNN

[0051] SEQ ID NO: 4 (UGT72E2): MHITKPHAAMFSSPGMGHVIPVIELGKRLSANNGFHVTVFVLETDAASAQSKFLNSTGVDIVKLPSPDIYGLVDPDDHVVTKIGVIMRAAVPALRSKIAAMHQKPTALIVDLFGTDALCLAKEFNMLSYVFIPTNARFLGVSIYYPNLDKDIKEEHTVQRNPLAIPGCEPVRFEDTLDAYLVPDEPVYRDFVRHGLAYPKADGILVNTWEEMEPKSLKSLLNPKLLGRVARVPVYPIGPL CRPIQSSETDHPVLDWLNEQPNESVLYISFGSGGCLSAKQLTELAWGLEQSQQRFVWVVRPPVDGSCCSEYVSANGGGTEDNTPEYLPEGFVSRTSDRGFVVPSWAPQAEILSHRAVGGF LTHCGWSSTLESVVGGVPMIAWPLFAEQNMNAALLSDELGIAVRLDDPKEDISRWKIEALVRKVMTEKEGEAMRRKVKKLRDSAEMSLSIDGGGLAHESLCRVTKECQRFLERVVDLSRGA

[0052] Examples 1-6, Comparative Examples 1-2: Evaluation of Alcohol Glycoside Activity The glycosyltransferases obtained above were used to compare alcohol glycosidation activity. The aromatic alcohol represented by general formula (1) (type and molar concentration listed in Table 1), 10 mM ascorbic acid derivative, 200 mM uridine diphosphate glucose (UDP-glucose), and 25 μg of the purified enzyme of each example were reacted in 500 μL of buffer (20 mM MES, pH 6.5 at 23°C) at 40°C for 3 hours with stirring. The molar ratio of the aromatic alcohol represented by general formula (1) to the ascorbic acid derivative (aromatic alcohol represented by general formula (1) / ascorbic acid derivative) was 10. The reaction was then terminated by adding 500 μL of methanol to the reaction system, followed by centrifugation (4°C, 1800 G, 30 min) to obtain the supernatant.

[0053] The supernatant was subjected to high performance liquid chromatography (HPLC) to quantitatively analyze the amount of the target product, alcohol glycoside. The HPLC analysis conditions were as follows: - HPLC analysis conditions - Apparatus: Nexera X2 (Shimadzu Corporation) Column: Shiseido Capecellpak C18 AQ, 5.0 μm, 4.6 mm × 250 mm Mobile phase: A: water (0.1% phosphoric acid), B: methanol (0.1% phosphoric acid) Time (min) / % B: 0 / 5, 15 / 5, 15 / 100, 25 / 100, 25 / 5 Injection volume: 2 μL Column temperature: 40°C Flow rate: 1.0 mL / min Detection wavelength: β-arbutin 280 nm, catechol monoglucoside 260 nm Retention time: β-arbutin 6.30 min, catechol monoglucoside 14.5 min

[0054] The alcohol glycosidation activity was calculated from the amount of alcohol glycoside produced by HPLC, the reaction time, and the amount of enzyme used, and evaluated according to the following criteria. The results are shown in the table below. Alcohol glycosidation activity (nkat / mg) = amount of alcohol glycoside produced (nmol) / reaction time (s) / amount of enzyme used (mg).

[0055] -Evaluation criteria- A: Alcohol glycosidation activity is 150 (nkat / mg) or more B: Alcohol glycosidation activity is 50 (nkat / mg) or more but less than 150 (nkat / mg) C: Alcohol glycosidation activity is 1 (nkat / mg) or more but less than 50 (nkat / mg) D: Alcohol glycosidation activity is less than 1 (nkat / mg)

[0056]

[0057] As shown in the above table, it was found that the production method of the Example was superior to the production method of the Comparative Example in alcohol glycoside activity under high concentration conditions.

[0058] The disclosure of Japanese Patent Application No. 2023-156508, filed on September 21, 2023, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for producing an alcohol glycoside, comprising contacting a glycosyltransferase with an aromatic alcohol represented by the following general formula (1), an ascorbic acid derivative, and a monosaccharide donor to form an alcohol glycoside: In general formula (1), R １ represents a hydroxy group, and n represents an integer of 1 or more and 5 or less.

2. The method for producing an alcohol glycoside according to claim 1, wherein the aromatic alcohol represented by the general formula (1) has a molar concentration of 50 mmol / L or more.

3. The method for producing an alcohol glycoside according to claim 1, wherein the molar ratio of the aromatic alcohol represented by the general formula (1) to the ascorbic acid derivative (aromatic alcohol represented by the general formula (1) / ascorbic acid derivative) is 5 or more and 20 or less.

4. The method for producing an alcohol glycoside according to claim 1, wherein the aromatic alcohol represented by the general formula (1) includes at least one selected from the group consisting of hydroquinone, resorcinol and catechol.

5. The method for producing an alcohol glycoside according to claim 1, wherein the monosaccharide donor comprises a uridine diphosphate sugar.

6. The method for producing an alcohol glycoside according to claim 1, wherein the glycosyltransferase has an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (UGT72B11), the amino acid sequence represented by SEQ ID NO: 2 (UGT72B1), or the amino acid sequence represented by SEQ ID NO: 3 (UGT73A10).

7. The method for producing an alcohol glycoside according to claim 6, wherein the glycosyltransferase has an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 (UGT72B11), the amino acid sequence represented by SEQ ID NO: 2 (UGT72B1), or the amino acid sequence represented by SEQ ID NO: 3 (UGT73A10).

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