JP2005312464A - Method for producing glycoside and transglucosylation product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a glycoside and a transglucosylation product by using γ-cyclodextrin glucanotransferase (γ-CGTase). <P>SOLUTION: The method for producing the glycoside and the transglucosylation product comprises treating a phenolic hydroxyl group-containing compound or a saccharide as a receptor and a proper sugar donor with γ-CGTase having wide receptor specificity and capable of acting in a wide pH region. For example, γ-CGTase produced by a Brevibacterium microorganism is used. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a glycoside and a glycosyl transfer product using γ-cyclodextrin glucanotransferase (hereinafter referred to as γ-CGTase). More specifically, a method for producing various glycosides or glycosyl transfer products by causing a compound or saccharide having various phenolic hydroxyl groups and γ-CGTase produced by Brevibacterium to act on a sugar donor. About.

  Conventionally, various glycosides have attracted attention as food materials, food additives, pharmaceuticals, and the like. That is, various glycosides are naturally present in small quantities, but are widely present, and are characterized by low toxicity despite having excellent physiological activity.

  For example, arbutin is known to have an antioxidant effect, antiallergic effect, melanin inhibitory action, etc., and is known as a highly safe topical skin preparation, and also has an action of maintaining a good balance of incense. It can be used as a fragrance-based ingredient in cosmetics. In addition, glycosides of catechol and resorcinol are known to prevent and treat skin pigmentation and the like and to prevent the occurrence of dandruff on the scalp.

  As described above, phenolic compounds have various physiological activities and are useful substances. By converting them into glycosides, problems such as side effects and solubility in water can be obtained without impairing their physiological activities. It is expected to improve the point.

  In addition, polyphenol glycosides are conventionally used not only as sweeteners, analgesics, laxatives, antimalarial agents, tonics, etc., but also useful as cosmetic ingredients that exhibit excellent whitening effects. Various methods for producing various glycosides using an intermolecular transfer reaction of an enzyme have been reported.

  For example, studies on amylases that transfer sugars to sugars and alcoholic hydroxyl groups [Amylase Symposium, Vol. 10, pp. 81-89 (1975)], phenols by allowing sucrose phosphorylase to act on phenolic compounds in the presence of sugar donors A method for producing glycosides (Japanese Patent Laid-Open No. 6-153976), a method for producing kojic acid glycosides by reacting kojic acid with sucrose phosphorylase in the presence of a sugar donor (Japanese Patent Laid-Open No. 6-056872), hydroxyfuranone A method for producing a furanone glycoside by reacting sucrose phosphorylase in the presence of a sugar donor (JP-A-6-135987), and a cyclodextrin synthase in the presence of a sugar donor such as cyclodextrin in ellagic acid For producing ellagic acid glycosides by reacting them (JP-A-5-331183), xylan and methylol placement A method for producing a xylo-oligosyl glycoside by allowing xylanase to act on a mixture of phenol derivatives (Japanese Patent Laid-Open No. 6-087880), and a method for producing a glycoside by allowing xylanase to act on a mixture of xylan and arylalkanol (Japanese Patent Laid-open No. 6-172403), a method for producing β-type polyphenol glycosides by reacting a sugar substrate with a polyphenol acceptor in the presence of cellulase (JP-A-6-284897), having no cyclodextrin synthesis ability and maltose resolution Various methods such as a method for producing a polyphenol glycoside using a novel enzyme (JP-A-6-284896) have been reported.

In addition, many transglycosylation reactions using various enzymes for sugar have been reported. For example, a method using β-galactosidase (JP-B-63-18457, JP-B-63-65301, JP-A-1-37991, JP-A-2-84191, JP-B-2-57902, JP-A-6-38785, etc.), β-D -A method using mannanase (Japanese Patent Laid-Open No. 5-155392), a method using β1,6-N-acetylglucosaminyltransferase (Japanese Patent Laid-Open No. 6-197756), a method using an enzyme derived from Bacillus cereus (Japanese Patent Laid-Open No. 6-298791) ), A method using CGTase derived from Bacillus stearothermophilus (Japanese Patent Publication No. 53-27791).
JP 61-274680 JP 62-25976 A Japanese Patent Laid-Open No. 6-11842 Japanese Patent Laid-Open No. 6-113843 New trend in cyclodextrins and derivatives, 25 pages (1991), published by Sante (Paris, France)

  However, in these methods of obtaining various glycosides and glycosyl transfer products, the receptor specificity of the enzyme used is a problem. Accordingly, it has been desired to develop a method capable of operating in a wide pH range.

  Therefore, the present inventors have conducted various studies to solve such problems, and as a result, a wider variety of receptor specificities can be exhibited by producing various glycosides using γ-CGTase. And the present invention was completed.

  That is, the present invention relates to a method for producing a glycoside or a glycosyl transfer product characterized in that a compound or saccharide having a phenolic hydroxyl group and γ-CGTase are allowed to act on a sugar donor.

  By using γ-CGTase having a wide receptor specificity, a glycoside of a compound having a phenolic hydroxyl group can be efficiently produced, and a glycosyl transfer product can also be produced. The process of the present invention can be carried out in a wide pH range, that is, neutral to alkaline.

Hereinafter, the present invention will be described in detail.
A method for producing a glycoside obtained by the present invention is as follows. 0.1-30%, preferably 0.5-5% of a compound having a phenolic hydroxyl group such as phenol, 1,3-dihydroxybenzene, 3-hydroxybenzyl alcohol, (+) catechin or kojic acid, malto-oligosaccharide, amylose, amylopectin or Γ-CGTase is added to a solution containing 0.1 to 30%, preferably 0.5 to 20%, of a sugar donor such as various starches, and 20 to 60 ° C, preferably 30 to 50 ° C, preferably 1 to 48 hours, preferably Let react for 10-24 hours to obtain a reaction solution. The pH of the reaction is preferably the optimum pH of the enzyme to be used, but can be determined in consideration of the solubility of the reaction product. For example, it is 3-11, preferably 5-9. A purified preparation of glycoside can be obtained by partitioning the reaction solution with various solvents and purifying by various chromatography.

  Moreover, the manufacturing method of the glycosyl transfer product obtained by this invention is as follows. Saccharides such as D-mannose and L-rhamnose 0.1 to 30%, preferably 1 to 10%, and sugar donors such as maltooligosaccharide, amylose, amylopectin or various starches, and 0.1 to 30%, preferably 1 to 10% Γ-CGTase is added to the contained solution and allowed to act at 20 to 60 ° C., preferably 30 to 50 ° C. for 1 to 48 hours, preferably 10 to 24 hours, to obtain a reaction solution. The pH of the reaction is good for the enzyme used, but can be determined in consideration of the solubility of the reaction product. For example, it is 3 to 11, preferably 5 to 9. A purified preparation of a glycosyl transfer product can be obtained from this reaction solution by partitioning with various solvents and purification by various chromatography.

  Examples of the compound having a phenolic hydroxyl group used in the present invention include Phenol, Benzyl alcohol, 1,2-Dihydroxybenzene, 1,3-Dihydroxybenzene, 1,4-Dihydroxybenzene, 1,2,3-Trihydroxybenzene, 1,3, 5-Trihydroxybenzene, 3-Hydroxybenzoic acid, 4-Hydroxybenzoic acid, 2-Hydroxybenzyl alcohol, 3-Hydroxybenzyl alcohol, 4-Hydroxybenzyl alcohol, Caffeic acid, Gallic acid, (+)-Catechin, (-)-Epigallocatechin gallate, Kojic acid Ascorbic acid, p-Nitrophenol, and the like. Examples of those that produce a glycoside better than CGTase derived from other sources (for example, Bacillus macerans and Bacillus stearothermophilus) include Phenol, 1,3-Dihydroxybenzene, 3-Hydroxybenzyl alcohol, (+) Catechin, Kojic acid and the like.

  Monosaccharides include D-glucose, D-galactose, D-mannose, D-fructose, D-glucosamine, D-arabinose, L-arabinose, D-xylose, D-ribose, D-fucose, L-fucose, L-rhamnose and the like can be mentioned, but examples of those with better transglycosylation products than CGTases derived from other sources (for example, Bacillus macerans and Bacillus stearothermophilus) include D-mannose and L -rhamnose.

  Γ-CGTase used in the present invention is an enzyme classified as cyclodextrin / glucanotransferase having an action of acting on starch to produce cyclodextrin, and α, β when acting on a starch solution. Among the γ-type cyclodextrins, it refers to an enzyme that mainly produces the γ-type. For example, Bacillus sp. AL6 CGTase (JP 61-274680), Bacillus sp. No. 313 CGTase (JP 62-25976) and Bacillus firmus 290-3 CGTase [New trend in cyclodextrins and derivatives 25 (1991), published by Sante (Paris, France)] and CGTase produced by Brevibacterium (Japanese Patent Laid-Open No. 6-11842) ), CGTase produced by Bacillus megaterium (JP-A-6-113843), more specifically, γ-CGTase derived from Brevibacterium sp. No. 9605 (FERM BP-4537) Etc. More preferably, γ-CGTase derived from Brevibacterium sp. No. 9605 can be used because of its wide working pH.

  The amount of γ-CGTase used in the present invention is 10 units or more, preferably 50 to 200 units, per gram of the total weight of the acceptor (compound or saccharide having a phenolic hydroxyl group) and the sugar donor. In the present invention, the activity of γ-CGTase was determined as follows.

  Activity measurement method: 0.05 ml of enzyme solution was added to 0.5 ml of a substrate [1.5% soluble starch, 0.1 M Atkins & Pantin buffer (pH 10.0)], and reacted at 40 ° C. for 30 minutes. Thereafter, 5 ml of 0.1N hydrochloric acid was added to stop the reaction, 0.5 ml was taken out, and 5 ml of iodine solution was added. The decrease in absorbance at 660 nm was measured. One unit was defined as the amount of enzyme that decreased the absorbance at 660 nm by 1% per minute under the present conditions.

  The present invention will be specifically described below with reference to test examples and examples, but the present invention is not limited to these examples.

Production of glycosides
Reaction solution containing 2.5 (w / v)% soluble starch (manufactured by Katayama Chemical), various receptors listed in Table 1 (compounds having a phenolic hydroxyl group) 2.5 (w / v)%, and 1.2 units of various CGTases (each pH buffer concentration 50 mM) was prepared and reacted at 40 ° C. for 20 hours. The pH 5.5 used an acetate buffer containing 5 mM CaCl ₂ , and the pH 7.0 and 9.0 used a borate buffer containing 5 mM CaCl ₂ . However, (+)-Catechin, (-)-Epigallocatechin gallate, and p-Nitrophenol were subjected to the reaction at concentrations of 0.5 (w / v)%. Also, CGTase is derived from Bacillus macerans (trade name: Contiszyme, Amano Pharmaceutical Co., Ltd.) (described as enzyme 1 in Table 1), derived from Bacillus stearothermophilus (produced by Hayashibara Seikagaku Corporation) ( In Table 1, described as enzyme 2) and from Brevibacterium sp. No. 9605 (FERM BP-4537) (manufactured by Amano Pharmaceutical Co., Ltd.) (described as enzyme 3 in Table 1) It was used.

  The transfer rate to various receptors was expressed as the ratio of the peak area of the transfer product to the total peak area by HPLC analysis. The HPLC analysis conditions were as follows: column: YMC ODS-AQ303 (manufactured by YMC), column temperature: 30 ° C., flow rate: 1.0 ml / min, and the following was used as the solvent.

(A): 20 (v / v)% methanol (prepared to pH 2.5 with phosphoric acid)
(B): 30 (v / v)% methanol (prepared to pH 2.5 with phosphoric acid)
(C): 40 (v / v)% methanol (prepared to pH 2.5 with phosphoric acid)
(D): 7.5 (v / v)% methanol (prepared to pH 2.5 with phosphoric acid)
(E): 50 mM KH ₂ PO ₄ (adjusted to pH 2.5 with phosphoric acid)
(F): 50 (v / v)% methanol (prepared to pH 2.5 with phosphoric acid)

  In Table 1, various receptors are indicated by the following abbreviations. In the table, “-” indicates that the production of glycoside could not be confirmed because the receptor was decomposed.

1,2-DHB: 1,2-Dihydroxybenzene (Catechol)
1,3-DHB: 1,3-Dihydroxybenzene (Resorcin)
1,4-DHB: 1,4-Dihydroxybenzene (Hydroquinone)
1,2,3-THB: 1,2,3-Trihydroxybenzene (Pyrogallol)
1,3,5-THB: 1,3,5-Trihydroxybenzene (Phloroglucinol)
3-HBAD: 3-Hydroxybenzoic acid
4-HBAD: 4-Hydroxybenzoic acid
2-HBAL: 2-Hydroxybenzyl alcohol
3-HBAL: 3-Hydroxybenzyl alcohol
4-HBAL: 4-Hydroxybenzyl alcohol
(-)-EGCg: (-)-Epigallocatechin gallate

  As is apparent from Table 1, when an enzyme derived from the genus Brevibacterium (enzyme 3) is used, it has a wide receptor specificity like CGTase derived from Bacillus stearothermophilus, especially phenol, For 1,3-dihydroxybenzene, 3-hydroxybenzyl alcohol, (+)-catechin and kojic acid, many glycosides are synthesized.

  In addition, enzymes derived from the genus Brevibacterium are capable of synthesizing glycosides even when neutral (pH 7.0) or alkaline (pH 9.0), and neutral or alkaline reactions in terms of receptor solubility. It is particularly useful when it is necessary.

Comparative Example According to Reaction pH In the same manner as in Example 1, 1,3-dihydroxybenzene, 1,3,5-trihydroxybenzene, (+)-catechin and kojic acid were used as receptors for enzyme 1, enzyme 2 and enzyme 3. The cases of pH 5.5 and pH 7.0 were compared. The results are shown in Table 2. In the table, the ratio a represents the production ratio of pH 5.5 and pH 7.0 in each enzyme, and the ratio b represents pH 5.5 and pH 7 when the amount of glycoside produced at each pH of enzyme 1 is 1. The production ratio when using each enzyme at 0.0 is shown.

  As is clear from Table 2, the enzyme derived from Brevibacterium (enzyme 3) shows a good glycoside formation rate even at pH 7.0, and in particular in the case of (+)-catechin, there is almost no influence of pH. It showed a high production rate.

Manufacture of glycosyltransferases 5 (w / v)% soluble starch (manufactured by Katayama Chemical), 200 μl of reaction solution containing 5 units (w / v)% of various monosaccharides (receptors) and 0.6 units of various CGTases (buffer at each pH) A liquid concentration of 30 mM) was prepared and reacted at 40 ° C. for 20 hours. The pH 5.5 used an acetate buffer containing 5 mM CaCl ₂ , and the pH 9.0 used a borate buffer containing 5 mM CaCl ₂ . Further, as CGTase, those derived from Bacillus macerans, Bacillus stearothermophilus and Brevibacterium sp. No. 9605 (FERM BP-4537) were used. After the reaction, the transglycosylation product was analyzed by TLC using 3 μl of the reaction solution. The result is shown in FIG. The symbols in the figure indicate the following cases.

R: oligosaccharide mixture
(Glucose, maltose, maltotriose, maltotetraose)
Bl: Blank (reacted in the same manner using water instead of enzyme solution)
M: When using CGTase derived from Bacillus macerans S: When using CGTase derived from Bacillus stearothermophilus B: When using γ-CGTase derived from Brevibacterium sp. No. 9605

(1) When no receptor is included
(2): D-glucose used as a receptor
(3): D-galactose is used as a receptor
(4): Uses D-mannose as a receptor
(5): Use D-fructose as a receptor
(6): Uses D-glucosamine as a receptor
(7): D-arabinose is used as a receptor
(8): L-arabinose is used as a receptor
(9): D-xylose is used as a receptor
(10): Use D-ribose as the acceptor
(11): Use D-fucose as a receptor
(12): L-fucose is used as a receptor
(13): L-rhamnose is used as a receptor

  When γ-CGTase derived from Brevibacterium sp. No. 9605 is used, the receptor specificity is wide as in the case of using CGTase derived from Bacillus stearothermophilus, especially D-mannose, L -When rhamnose was used as a receptor, many transfer products were synthesized.

It is a figure which shows the TCL result of Example 3.

Claims (5)

  A method for producing a glycoside, comprising reacting a compound having a phenolic hydroxyl group and a sugar donor with γ-cyclodextrin / glucanotransferase.   A method for producing a glycosyl transfer product, which comprises reacting a saccharide and a sugar donor with γ-cyclodextrin / glucanotransferase.   The production method according to claim 1 or 2, wherein the method is operated under neutral or alkaline conditions.   The production method according to claim 1 or 2, wherein the γ-cyclodextrin glucanotransferase is an enzyme derived from the genus Brevibacterium. The process according to claim 4, wherein the microorganism belonging to the genus Brevibacterium is Brevibacterium sp. No. 9605 (FERM BP-4537).

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