JPH06284897A - Production of beta-form polyphenol glycoside - Google Patents
Production of beta-form polyphenol glycosideInfo
- Publication number
- JPH06284897A JPH06284897A JP26132992A JP26132992A JPH06284897A JP H06284897 A JPH06284897 A JP H06284897A JP 26132992 A JP26132992 A JP 26132992A JP 26132992 A JP26132992 A JP 26132992A JP H06284897 A JPH06284897 A JP H06284897A
- Authority
- JP
- Japan
- Prior art keywords
- polyphenol
- cellulase
- beta
- glycoside
- polyphenol glycoside
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】この発明は、セルロース、セロオ
リゴ糖およびカルボキシメチルセルロース等の糖基質を
分解するセルラーゼを用いるβ型ポリフェノール配糖体
の製法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing β-type polyphenol glycosides using a cellulase which decomposes sugar substrates such as cellulose, cellooligosaccharides and carboxymethylcellulose.
【０００２】[0002]
【従来の技術】ポリフェノール配糖体は、従来から、甘
味料、鎮痛剤、下剤、抗マラリヤ剤および強壮剤等とし
て利用されているだけでなく、優れた美白効果を発揮す
る化粧品の配合成分としても利用できる(特開平３−３
４１５１号明細書参照)有用な化合物であり、本件出願
人は先に、サイクロデキストリン合成能とマルトース分
解能を有さない新規な酵素を用いるポリフェノール配糖
体の製法(特願平４−２７９２６号明細書参照)、および
糖化型アミラーゼを用いるポリフェノール配糖体の製法
(特願平４−１２３１０３)を提供した。しかしながら、
該ポリフェノール配糖体の糖の結合様式はα型であり、
該方法では天然界に多く存在するβ型を製造することが
できない。例えば、次式で表わされるハイドロキノン−
０−β−Ｄ−グルコピラノシド(アルブチン)は美白剤と
して応用されているが、該方法では製造することができ
ない:2. Description of the Related Art Polyphenol glycosides have hitherto been used as sweeteners, analgesics, laxatives, antimalarial agents, tonic agents, etc., and also as a blending component of cosmetics which exert an excellent whitening effect. Can also be used (Japanese Patent Laid-Open No. 3-3
This is a useful compound, and the applicant of the present invention has previously described a method for producing a polyphenol glycoside using a novel enzyme having no cyclodextrin synthesizing ability and maltose decomposing ability (Japanese Patent Application No. 4-27926). ), And a method for producing polyphenol glycosides using saccharified amylase
(Japanese Patent Application No. 4-123103). However,
The binding mode of sugar of the polyphenol glycoside is α-type,
This method cannot produce β type, which is often present in the natural world. For example, hydroquinone represented by the following formula
0-β-D-Glucopyranoside (Arbutin) has been applied as a whitening agent but cannot be produced by the method:
【０００３】[0003]
【化１】 [Chemical 1]
【０００４】一方、化学合成によりβ型ポリフェノール
配糖体を作る方法は既に知られているが、合成操作が多
段階におよぶだけでなく、合成過程で多くの副産物が生
じ、目的物の分離および精製は非常に困難である。On the other hand, a method for producing a β-type polyphenol glycoside by chemical synthesis has already been known, but not only the synthetic operation involves multiple steps, but also many by-products are generated in the synthetic process to separate the desired product and Purification is very difficult.
【０００５】[0005]
【発明が解決しようとする課題】この発明は、さらに別
異の酵素を用いて、β型ポリフェノール配糖体を一段階
で選択的に製造する方法を提供するためになされたもの
である。The present invention has been made to provide a method for selectively producing a β-type polyphenol glycoside in one step by using a different enzyme.
【０００６】[0006]
【課題を解決するための手段】即ちこの発明は、セルラ
ーゼがβ型ポリフェノール配糖体合成能を有するという
知見に基づいてなされたものであって、その要旨はセル
ラーゼの存在下において、糖基質とポリフェノール受容
体を反応させることを特徴とするβ型ポリフェノール配
糖体の製法に存する。Means for Solving the Problems That is, the present invention was made based on the finding that cellulase has β-type polyphenol glycoside synthesizing ability. It exists in a method for producing a β-type polyphenol glycoside characterized by reacting a polyphenol receptor.
【０００７】本発明に使用するセルラーゼは自体公知の
酵素であるが、該酵素がポリフェノール配糖体合成能を
有するということは全く知られていなかった。この種の
セルラーゼとしては、アスペルギルス属またはトリコデ
ルマ属の微生物、例えば、トリコデルマ・ビリデ、トリ
コデルマ・コニンギ、トリコデルマ・ロンギブラチアタ
ム、トリコデルマ・リーセイ、アスペルギルス・ニーガ
ー、アスペルギルス・サイトイ、アスペルギルス・フラ
ブス、アスペルギルス・オリーゼまたはアスペルギルス
・フミガトウス等の微生物から生産される上記特性を有
するセルラーゼが例示される。The cellulase used in the present invention is an enzyme known per se, but it has not been known at all that the enzyme has a polyphenol glycoside synthesizing ability. Examples of this type of cellulase include microorganisms of the genus Aspergillus or Trichoderma, for example, Trichoderma viride, Trichoderma coningi, Trichoderma longibrachiatum, Trichoderma reesei, Aspergillus niger, Aspergillus cytoii, Aspergillus flavus, Aspergillus oryzae. Alternatively, a cellulase produced from a microorganism such as Aspergillus fumigatus and having the above characteristics is exemplified.
【０００８】上記の微生物を用いるセルラーゼの調製は
自体公知の方法、例えば、岡田らの方法[ザ・ジャーナ
ル・オブ・バイオケミストリー(Ｊ．Ｂiochem．)、第８
０巻、第９１３頁〜第９２２頁(１９７６年)]および松
村らの方法[発酵工業会誌、第４１巻、第１５８頁〜第
１６３頁(１９６３年)]等に準拠して行えばよい。The preparation of cellulase using the above-mentioned microorganisms is a method known per se, for example, the method of Okada et al. [The Journal of Biochemistry (J. Biochem.), No. 8].
0, pp. 913 to 922 (1976)] and the method of Matsumura et al. [Journal of Fermentation Industry, Vol. 41, 158 to 163 (1963)] and the like.
【０００９】上記の特性を有するセルラーゼは、広範囲
の糖基質を加水分解し、種々のポリフェノール類にグル
コースを転移させ、これによって多種多様なβ型ポリフ
ェノール配糖体が得られる。この種の糖基質およびポリ
フェノール受容体としては下記のものが例示される:糖基質 ;セルロース、カルボキシメチルセルロース、セ
ロオリゴ糖(Ｇ2以上)。ポリフェノール受容体 ;カテキン、ハイドロキノン、レ
ゾルシノール、α−レゾルシル酸、γ−レゾルシル酸、
フロログルシノール、サリチルアルコール、p−ハイド
ロキシベンジルアルコール、m−ハイドロキシベンジル
アルコール。The cellulase having the above-mentioned properties hydrolyzes a wide range of sugar substrates and transfers glucose to various polyphenols, whereby various β-type polyphenol glycosides can be obtained. Examples of sugar substrates and polyphenol acceptors of this type include the following: sugar substrates ; cellulose, carboxymethyl cellulose, cellooligosaccharides (G 2 and above). Polyphenol receptor ; catechin, hydroquinone, resorcinol, α-resorcylic acid, γ-resorcylic acid,
Phloroglucinol, salicyl alcohol, p-hydroxybenzyl alcohol, m-hydroxybenzyl alcohol.
【００１０】セルロース等の糖基質およびカテキン等の
ポリフェノール受容体を、上述の酵素の存在下で反応さ
せる方法は、通常、酢酸緩衝液等の緩衝液を用いて反応
系のpＨを約３〜９に調整し、約１０〜６０℃で約３〜
１００時間おこなう。反応溶媒としては、水、メタノー
ル／水(５〜５０体積％)、エタノール／水(５〜５０体
積％)、酢酸エチル／水(１０〜８０体積％)、アセトニ
トリル／水(２０〜８０体積％)等が例示される。また、
使用する酵素を不溶性担体に固定化することにより、製
造したβ型ポリフェノール配糖体から酵素を除く操作を
省略することもできる。In the method of reacting a sugar substrate such as cellulose and a polyphenol receptor such as catechin in the presence of the above-mentioned enzyme, a pH of the reaction system is usually about 3 to 9 using a buffer such as an acetate buffer. To about 3 to 60 at about 10 to 60 ℃
Do it for 100 hours. As the reaction solvent, water, methanol / water (5 to 50% by volume), ethanol / water (5 to 50% by volume), ethyl acetate / water (10 to 80% by volume), acetonitrile / water (20 to 80% by volume) ) Etc. are illustrated. Also,
By immobilizing the enzyme to be used on an insoluble carrier, the operation of removing the enzyme from the produced β-type polyphenol glycoside can be omitted.
【００１１】[0011]
【実施例】以下、本発明を実施例によって説明する。実施例１ (ポリフェノール配糖体合成活性測定法) ハイドロキノンの酢酸エチル溶液(０．５Ｍ)０．５mlお
よびカルボキシメチルセルロース(ナカライテスク株式
会社製)３w／v％、酢酸緩衝液(pＨ４．５)５０mＭおよ
びセルラーゼＴ「アマノ」(天野製薬株式会社製)３０mg／
mlを含有する水溶液０．５mlを蓋付試験管(２ml)に入
れ、該試験管を回転数２８０rpmの条件下において、４
０℃で１８時間振盪させた。静置後、水性層を下記の条
件下での薄層クロマトグラフィー分析に付し、酵素のポ
リフェノール配糖体合成活性を確認した。 薄層 :メルク社製シリカゲル６０Ｆ２５４ガラスプ
レート 展開溶媒:酢酸エチル／酢酸／水＝３／１／１(体積比) 検出 :３３v／v％硫酸／メタノール混合液を薄層に
噴霧後、該薄層を１２０℃で１０分間加熱する。 Ｒf値 :０．５４〜０．６２(カルボキシメチルセルロ
ースの分解物のＲf値は０．２４以下である)EXAMPLES The present invention will be described below with reference to examples. Example 1 (Method for measuring polyphenol glycoside synthesis activity) 0.5 ml of an ethyl acetate solution of hydroquinone (0.5 M), 3 w / v% of carboxymethyl cellulose (manufactured by Nacalai Tesque, Inc.), 50 mM acetate buffer (pH 4.5) And Cellulase T "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) 30 mg /
0.5 ml of an aqueous solution containing ml was placed in a test tube with a lid (2 ml), and the test tube was placed under the condition of a rotation speed of 280 rpm to
Shake at 0 ° C. for 18 hours. After standing, the aqueous layer was subjected to thin layer chromatography analysis under the following conditions to confirm the polyphenol glycoside synthesis activity of the enzyme. Thin layer: Silica gel 60F254 glass plate manufactured by Merck & Co., Inc. Developing solvent: ethyl acetate / acetic acid / water = 3/1/1 (volume ratio) Detection: 33 v / v% sulfuric acid / methanol mixed solution is sprayed onto the thin layer, and then the thin layer Is heated at 120 ° C. for 10 minutes. Rf value: 0.54 to 0.62 (Rf value of decomposed product of carboxymethylcellulose is 0.24 or less)
【００１２】実施例２〜９ (受容体特異性) ハイドロキノンの代わりに、カテキン、カテコール、レ
ゾルシノール、α−レゾルシル酸、フロログルシノー
ル、サリチルアルコール、p−ハイドロキシベンジルア
ルコールまたはm−ハイドロキシベンジルアルコールを
使用する以外は、実施例１の手順に準拠して、本発明に
用いる酵素のポリフェノール配糖体合成活性を確認し
た。 Examples 2 to 9 (Receptor Specificity) Catechin, catechol, resorcinol, α-resorcylic acid, phloroglucinol, salicyl alcohol, p-hydroxybenzyl alcohol or m-hydroxybenzyl alcohol were used instead of hydroquinone. Other than that, according to the procedure of Example 1, the polyphenol glycoside synthesizing activity of the enzyme used in the present invention was confirmed.
【００１３】実施例１０(アルブチンの調製) セルラーゼＴ「アマノ」(天野製薬株式会社製)３０mg／m
l、ハイドロキノン７w／v％、濾紙粉末５w／v％および
酢酸緩衝液(pＨ４．５)５０mＭから成る混合液１００ml
を三角フラスコ(２００ml)内に入れ、該フラスコを回転
数３００rpmの条件下において、４０℃で９６時間振盪
させた。反応液をロータリーエバポレーターを用いる減
圧濃縮乾燥処理に付した後、残渣を水１０mlに溶解さ
せ、該水溶液を下記の条件下でのカラムクロマトグラフ
ィー処理に付し、溶離液体積４４０〜４８０mlで反応生
成物２を、同体積４８０〜５４０mlで反応生成物１を溶
離させた。 カラム:内径２．６cm×長さ９１cm 充填剤:バイオゲルp−２(４００メッシュ) 溶離液:５％(v／v)エタノール水溶液 Example 10 (Preparation of arbutin) Cellulase T "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.) 30 mg / m
l, hydroquinone 7w / v%, filter paper powder 5w / v% and acetate buffer (pH 4.5) 50mM mixed solution 100ml
Was placed in an Erlenmeyer flask (200 ml), and the flask was shaken at 40 ° C. for 96 hours under the condition of a rotation speed of 300 rpm. The reaction solution was concentrated and dried under reduced pressure using a rotary evaporator, the residue was dissolved in 10 ml of water, and the aqueous solution was subjected to column chromatography under the following conditions to produce a reaction mixture with an eluent volume of 440 to 480 ml. Reaction product 1 was eluted with the same volume of 480 to 540 ml. Column: Inner diameter 2.6 cm x length 91 cm Packing material: Biogel p-2 (400 mesh) Eluent: 5% (v / v) ethanol aqueous solution
【００１４】得られた画分を、それぞれロータリーエバ
ポレーターを用いて減圧濃縮乾固された後、さらに真空
乾燥機を用いて乾燥処理を行って、白色粉末として生成
物１を８mg、生成物２を５mg得た。該生成物は下記の条
件下での薄層クロマトグラフィーにおいて単一であっ
た。 (生成物１のＲf値:０．６２、生成物２のＲf値:０．５
４): 薄層 :メルク社製シリカゲル６０Ｆ２５４ガラスプ
レート 展開溶媒:酢酸エチル／酢酸／水＝３:１:１(体積比) 検出 :３３v／v％硫酸／メタノール混合液を薄層に
噴霧後、該薄層を１２０℃で １０分間熱
する。1 Ｈ−ＮＭＲ分析の結果、該生成物１および２をそれぞ
れハイドロキノン−０−β−Ｄ−グルコピラノシド(ア
ルブチン)、ハイドロキノン−０−β−セロビオシド(ア
ルブチン−０−β−Ｄ−グルコピラノシド)と同定し
た。The obtained fractions were concentrated to dryness under reduced pressure using a rotary evaporator, and then dried using a vacuum dryer to obtain 8 mg of the product 1 as a white powder and 2 mg of the product 2. 5 mg was obtained. The product was single in thin layer chromatography under the following conditions. (Rf value of product 1: 0.62, Rf value of product 2: 0.5
4): Thin layer: Silica gel 60F254 glass plate manufactured by Merck & Co., Inc. Developing solvent: ethyl acetate / acetic acid / water = 3: 1: 1 (volume ratio) Detection: 33v / v% sulfuric acid / methanol mixed solution was sprayed on the thin layer, The thin layer is heated at 120 ° C. for 10 minutes. As a result of 1 H-NMR analysis, the products 1 and 2 were identified as hydroquinone-0-β-D-glucopyranoside (arbutin) and hydroquinone-0-β-cellobioside (arbutin-0-β-D-glucopyranoside), respectively. .
【００１５】[0015]
【発明の効果】本発明によれば、セルラーゼ、例えば、
アスペルギルス属またはトリコデルマ属等の微生物が生
産するセルラーゼを利用することによって、広範囲の糖
基質とポリフェノール受容体を原料として、特に医薬や
化粧品等の配合成分として有用な多種多様なβ型ポリフ
ェノール配糖体を効率よく製造することができる。According to the present invention, a cellulase, for example,
By using cellulases produced by microorganisms such as Aspergillus or Trichoderma, a wide variety of β-polyphenol glycosides that are useful as a raw material from a wide range of sugar substrates and polyphenol acceptors, especially as a compounding component of medicines, cosmetics, etc. Can be manufactured efficiently.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 (Ｃ１２Ｐ 19/44 Ｃ１２Ｒ 1:67） (Ｃ１２Ｐ 19/44 Ｃ１２Ｒ 1:69） (72)発明者 西野 豊和 大阪府寝屋川市下木田町14番５号 倉敷紡 績株式会社技術研究所内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification number Office reference number FI technical display location (C12P 19/44 C12R 1:67) (C12P 19/44 C12R 1:69) (72) Inventor Toyowa Nishino 14-5 Shimokita-cho, Neyagawa-shi, Osaka Kurashiki Spinning Co., Ltd.
Claims (4)
ポリフェノール受容体を反応させることを特徴とするβ
型ポリフェノール配糖体の製法。1. A β which is characterized by reacting a sugar substrate with a polyphenol receptor in the presence of cellulase.
Of producing type polyphenol glycosides.
リコデルマ属の微生物が生産するセルラーゼである請求
項１記載の方法。2. The method according to claim 1, wherein the cellulase is a cellulase produced by a microorganism of the genus Aspergillus or the genus Trichoderma.
リゴ糖またはカルボキシメチルセルロースである請求項
１記載の方法。3. The method according to claim 1, wherein the sugar substrate is cellulose, G 2 or higher cellooligosaccharide or carboxymethyl cellulose.
ドロキノン、レゾルシノール、α−レゾルシル酸、γ−
レゾルシル酸、フロログルシノール、サリチルアルコー
ル、p−ハイドロキシベンジルアルコールまたはm−ハイ
ドロキシベンジルアルコールである請求項１記載の方
法。4. The polyphenol receptor is catechin, hydroquinone, resorcinol, α-resorcylic acid, γ-.
The method according to claim 1, which is resorcylic acid, phloroglucinol, salicyl alcohol, p-hydroxybenzyl alcohol or m-hydroxybenzyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26132992A JPH0736759B2 (en) | 1992-09-30 | 1992-09-30 | Method for producing β-type polyphenol glycoside |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26132992A JPH0736759B2 (en) | 1992-09-30 | 1992-09-30 | Method for producing β-type polyphenol glycoside |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06284897A true JPH06284897A (en) | 1994-10-11 |
| JPH0736759B2 JPH0736759B2 (en) | 1995-04-26 |
Family
ID=17360303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26132992A Expired - Lifetime JPH0736759B2 (en) | 1992-09-30 | 1992-09-30 | Method for producing β-type polyphenol glycoside |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0736759B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005038038A1 (en) * | 2003-10-16 | 2005-04-28 | Nichirei Foods Inc. | Process for producing ascorbic acid glycoside |
| WO2008088047A1 (en) * | 2007-01-19 | 2008-07-24 | Suntory Holdings Limited | Method for glycosylation of flavonoid |
-
1992
- 1992-09-30 JP JP26132992A patent/JPH0736759B2/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005038038A1 (en) * | 2003-10-16 | 2005-04-28 | Nichirei Foods Inc. | Process for producing ascorbic acid glycoside |
| WO2008088047A1 (en) * | 2007-01-19 | 2008-07-24 | Suntory Holdings Limited | Method for glycosylation of flavonoid |
| JP2008174507A (en) * | 2007-01-19 | 2008-07-31 | Suntory Ltd | Method for glycosidizing flavonoid |
| CN101605905A (en) * | 2007-01-19 | 2009-12-16 | 三得利控股株式会社 | The glucosides method of flavonoid class |
| US9364492B2 (en) | 2007-01-19 | 2016-06-14 | Suntory Holdings Limited | Method for glycosylation of flavonoid compounds |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0736759B2 (en) | 1995-04-26 |
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