CN101280332A - Enzymatic preparation of alpha-galactosyl-beta-cyclodextrin - Google Patents
Enzymatic preparation of alpha-galactosyl-beta-cyclodextrin Download PDFInfo
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- CN101280332A CN101280332A CNA2008101002719A CN200810100271A CN101280332A CN 101280332 A CN101280332 A CN 101280332A CN A2008101002719 A CNA2008101002719 A CN A2008101002719A CN 200810100271 A CN200810100271 A CN 200810100271A CN 101280332 A CN101280332 A CN 101280332A
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- cyclodextrin
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- galactosyl
- melibiose
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Abstract
The invention relates to a preparation method for the enzyme method of Alpha-galactosyl-beta-cyclodextrin, which relates to the cyclodextrin echnology field. The method takes beta-cyclodextrin and melibiose as the raw material, and the branching cyclodextrin of alpha-galactosyl-beta-cyclodextrin is produced after the procedures such as solubilization, synthesis, rough separation, purification and drying, etc., through the reverse synthesis action of alpha-galactosidase. The obtained alpha-galactosyl-beta-cyclodextrin has high purity, good water solubility and steady property, and the preparation method is provided with intensive application and study value.
Description
Technical field
A kind of enzymatic-process preparation method of alpha-galactosyl-beta-cyclodextrin relates to the cyclodextrin technical field.
Background technology
Cyclodextrin (Cyclodextrin) is the ring type oligose that is formed by connecting with α-1,4 glycosidic link by several glucosyl residues.The ring texture of cyclodextrin provides a hydrophobic internal cavities, and free hydroxyl is positioned at the ring outside, makes cyclodextrin have interior hydrophobic, outer hydrophilic special property.This special property of cyclodextrin has in fields such as pharmacy, chemical industry, food in recent years and studies widely and use.More common cyclodextrin be the α that forms by 6,7 and 8 glucosyl residues respectively-, β-and γ-Huan Hujing.The beta-cyclodextrin production technique that wherein contains 7 glucosyl residues is simple, and cost is lower, is at present industrial unique energy mass production and the wider cyclodextrin product of application.
But beta-cyclodextrin has bad water-solubility, has restricted its further application in every respect.At present, the modification by beta-cyclodextrin can reach the water miscible purpose of increase.Generally by chemical method and two kinds of methods of enzyme process, with respect to the chemical method modification, enzyme modification then has advantages such as specificity is strong, security height in the modification of beta-cyclodextrin.
The present invention is a raw material with beta-cyclodextrin and melibiose, and the reverse Synthesis by alpha-galactosidase prepares alpha-galactosyl-beta-cyclodextrin.This branching cd has higher water-soluble, and the semi-lactosi in its branch has special identification receptor in animal tissues.Therefore, have broad application prospects.
Summary of the invention
The purpose of this invention is to provide a kind of is raw material with beta-cyclodextrin and melibiose, by the reverse Synthesis of alpha-galactosidase, prepares the method for alpha-galactosyl-beta-cyclodextrin.This branching cd has better water solubility, and its semi-lactosi side chain has special identification receptor in animal tissues.Have broad application prospects.
Technical scheme of the present invention: a kind of enzymatic-process preparation method of alpha-galactosyl-beta-cyclodextrin, with beta-cyclodextrin and melibiose is raw material, reverse Synthesis by alpha-galactosidase, through operations such as dissolving, synthetic, rough segmentation, purifying, dryings, prepare alpha-galactosyl-beta-cyclodextrin.Preparation technology is as follows:
(1) raw material: with beta-cyclodextrin and melibiose is raw material, and beta-cyclodextrin and melibiose mol ratio are 1: 1-2, alpha-galactosidase 20～30U/g melibiose.
(2) dissolving: dry powder beta-cyclodextrin and melibiose raw mix are put into the container that fills 25～50mmol/L, pH 5～7 acetate buffer solutions, stirring and dissolving, consumption is beta-cyclodextrin 0.1～0.2mol/L, melibiose 0.1～0.2mol/L drips alpha-galactosidase liquid in mixed system.Because water-soluble relatively poor, the beta-cyclodextrin in the mixed system can not dissolve fully, presents hypersaturated state.
(3) synthetic: as container to be placed constant temperature oscillator, carry out building-up reactions, 20～30 ℃ of temperature of reaction, reaction times 36～72h, hunting speed 150～200r/min.
(4) rough segmentation: building-up reactions is put into boiling water with container and is soaked 2～3min after finishing, and makes the abundant deactivation of alpha-galactosidase.It is centrifugal that mixed solution after the deactivation is put into centrifuge tube.Centrifugal speed 2 000～3 000r/min, centrifugation time 2～3min.Supernatant liquor is got in centrifugal end.
(5) purifying: the centrifugal supernatant liquor that obtains is carried out separation and purification with preparative high performance liquid chromatography (HPLC).Prep C18 OBD (5 μ m, 19 * 150mm) posts, light scattering detector (SEDEX75FRANCE), Waters 1525 Binary HPLC pumps, sample introduction 1～1.5mL, 20～25 ℃ of column temperatures, the methanol aqueous solution of moving phase 1%～3%, flow velocity 2.0～3.0mL/min, splitting ratio 35: 1～30: 1.Character pair peak a collects sample solution, obtains alpha-galactosyl-beta-cyclodextrin.
(6) drying: the alpha-galactosyl-beta-cyclodextrin solution lyophilize that will obtain through the preparative high performance liquid chromatography separation and purification, temperature-40 ℃, vacuum tightness≤12Pa, time of drying, 24～48h obtained the alpha-galactosyl-beta-cyclodextrin powder.
Beneficial effect of the present invention: with beta-cyclodextrin and melibiose is raw material, prepares alpha-galactosyl-beta-cyclodextrin.This branching cd has higher water-soluble, and its semi-lactosi side chain has special identification receptor in animal tissues.Therefore, have broad application prospects.The transformation efficiency of the alpha-galactosyl-beta-cyclodextrin that the present invention makes is 25～30% (calculating with the beta-cyclodextrin that transforms), and purity is 90～95%, has higher water-soluble.This product has purity height, good water solubility, characteristics such as stable in properties.Beta-cyclodextrin and melibiose that the present invention adopts obtain easily, and methods such as the dissolving of adopting in the technology, synthetic, rough segmentation, purifying, drying are all ordinary method, and are simple and practical, and non-pollution discharge is environmentally friendly.
Description of drawings
Fig. 1: preparation high performance liquid phase (HPLC) collection of illustrative plates.
Embodiment
Raw material: beta-cyclodextrin 0.1mol/L (2g), melibiose 0.16mol/L (1g), alpha-galactosidase 25U.Complete processing: through dissolving (30mmol/L acetate buffer solution 20mL, pH6, beta-cyclodextrin supersaturation), synthetic (25 ℃ of temperature of reaction, reaction times 48h, hunting speed 200r/min), (boiling water soaks 2min, centrifugal speed 2500r/min in rough segmentation, centrifugation time 2min), purifying (sample introduction 1.5mL, 25 ℃ of column temperatures, the methanol aqueous solution of moving phase 1%, flow velocity 2.0mL/min, splitting ratio 30: 1), dry (temperature-40 ℃, vacuum tightness≤12Pa, time of drying 36h) make product.The transformation efficiency that makes alpha-galactosyl-beta-cyclodextrin is 25%, and purity is 95%.
Claims (1)
1, a kind of enzymatic-process preparation method of alpha-galactosyl-beta-cyclodextrin, it is characterized in that with beta-cyclodextrin and melibiose be raw material, by the reverse Synthesis of alpha-galactosidase,, prepare alpha-galactosyl-beta-cyclodextrin through dissolving, synthetic, rough segmentation, purifying, drying process; Technology is as follows:
(1) raw material: with beta-cyclodextrin and melibiose is raw material, and beta-cyclodextrin and melibiose mol ratio are 1: 1-2, alpha-galactosidase 20～30U/g melibiose;
(2) dissolving: dry powder beta-cyclodextrin and melibiose raw mix are put into the container that fills 25～50mmol/L, pH 5～7 acetate buffer solutions, stirring and dissolving, consumption is beta-cyclodextrin 0.1～0.2mol/L, melibiose 0.1～0.2mol/L drips alpha-galactosidase liquid in mixed system; Because water-soluble relatively poor, the beta-cyclodextrin in the mixed system can not dissolve fully, presents hypersaturated state;
(3) synthetic: as container to be placed constant temperature oscillator, carry out building-up reactions, 20～30 ℃ of temperature of reaction, reaction times 36～72h, hunting speed 150～200r/min;
(4) rough segmentation: building-up reactions is put into boiling water with container and is soaked 2～3min after finishing, and makes the abundant deactivation of alpha-galactosidase, and it is centrifugal that the mixed solution after the deactivation is put into centrifuge tube, centrifugal speed 2,000～3,000r/min, centrifugation time 2～3min, supernatant liquor is got in centrifugal end;
(5) purifying: the centrifugal supernatant liquor that obtains is carried out separation and purification with preparative high performance liquid chromatography, PrepC18 OBD 5 μ m, 19 * 150mm post, light scattering detector SEDEX75 FRANCE, Waters 1525 Binary HPLC pumps, sample introduction 1～1.5mL, 20～25 ℃ of column temperatures, the methanol aqueous solution of moving phase 1%～3%, flow velocity 2.0～3.0mL/min, splitting ratio 35: 1～30: 1; Character pair peak a collects sample solution, obtains alpha-galactosyl-beta-cyclodextrin solution;
(6) drying: the alpha-galactosyl-beta-cyclodextrin solution lyophilize that will obtain through the preparative high performance liquid chromatography separation and purification, temperature-40 ℃, vacuum tightness≤12Pa, time of drying, 24～48h obtained the alpha-galactosyl-beta-cyclodextrin powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101002719A CN101280332A (en) | 2008-05-09 | 2008-05-09 | Enzymatic preparation of alpha-galactosyl-beta-cyclodextrin |
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| CNA2008101002719A CN101280332A (en) | 2008-05-09 | 2008-05-09 | Enzymatic preparation of alpha-galactosyl-beta-cyclodextrin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994562A (en) * | 2012-10-10 | 2013-03-27 | 贵州泰然环境科技有限责任公司 | Technology for preparing methane through vinasse fermentation |
| CN108003198A (en) * | 2017-11-15 | 2018-05-08 | 昆明理工大学 | A kind of bonded open loop Cucurbituril of D- galactolipins and application |
| CN115428936A (en) * | 2022-08-12 | 2022-12-06 | 上海统益生物科技有限公司 | Masking agent for improving bitter taste of food and preparation method and application thereof |
-
2008
- 2008-05-09 CN CNA2008101002719A patent/CN101280332A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994562A (en) * | 2012-10-10 | 2013-03-27 | 贵州泰然环境科技有限责任公司 | Technology for preparing methane through vinasse fermentation |
| CN108003198A (en) * | 2017-11-15 | 2018-05-08 | 昆明理工大学 | A kind of bonded open loop Cucurbituril of D- galactolipins and application |
| CN108003198B (en) * | 2017-11-15 | 2021-06-18 | 昆明理工大学 | D-galactose-bonded ring-opening cucurbituril and application thereof |
| CN115428936A (en) * | 2022-08-12 | 2022-12-06 | 上海统益生物科技有限公司 | Masking agent for improving bitter taste of food and preparation method and application thereof |
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Open date: 20081008 |