阅读说明：本技术 一种合成l-抗坏血酸-2-葡萄糖苷的方法 (method for synthesizing L-ascorbic acid-2-glucoside ) 是由 王珂 周友超 陈修 陈斌 朱东来 于 2019-10-30 设计创作，主要内容包括：本发明属于生化合成技术领域,具体公开了一种合成L-抗坏血酸-2-葡萄糖苷的方法。该合成方法采用5,6-O-异丙叉基-L-抗坏血酸为起始原料,先反应制得5,6-O-异丙叉基-L-抗坏血酸-2-葡萄糖苷,反应得到的反应液用强酸性阳离子交换树脂进行分离,5,6-O-异丙叉基-L-抗坏血酸-2-葡萄糖苷即可水解为L-抗坏血酸-2-葡萄糖苷,然后再进行结晶、干燥,即得到L-抗坏血酸-2-葡萄糖苷产品,结晶后剩余的料液循环使用。本发明的合成方法有效避免了副产物生成,降低了后续分离纯化难度,提高了L-抗坏血酸-2-葡萄糖苷产品的纯度和收率。(The invention belongs to the technical field of biochemical synthesis, and particularly discloses methods for synthesizing L-ascorbic acid-2-glucoside, wherein 5, 6-O-isopropylidene-L-ascorbic acid is used as a starting material, 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside is prepared by reaction, reaction liquid obtained by the reaction is separated by using strong acid cation exchange resin, 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside can be hydrolyzed into L-ascorbic acid-2-glucoside, crystallization and drying are carried out, so that an L-ascorbic acid-2-glucoside product is obtained, and residual feed liquid after crystallization is recycled.)

1, methods for synthesizing L-ascorbic acid-2-glucoside, comprising the steps of:

(1) taking 5, 6-O-isopropylidene-L-ascorbic acid as a reaction raw material, adding a glucose group donor, and carrying out a biotransformation reaction under the action of glycosyltransferase;

(2) adding glucoamylase into the reaction liquid obtained after the reaction in the step (1) for hydrolysis reaction to prepare 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside;

(3) and (3) separating the reaction liquid containing the 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside obtained in the step (2) by adopting a strong acid cation exchange resin, crystallizing the separation liquid to separate out an L-ascorbic acid-2-glucoside product, and returning the residual feed liquid after the product is separated out to the step (1) for recycling.

2. The method for synthesizing L-ascorbic acid-2-glucoside according to claim 1, wherein the amount of the glucose-based donor is 50 to 60% of the mass of the 5, 6-O-isopropylidene-L-ascorbic acid; and/or, the glycosyltransferase is used in an amount of: adding 650-700U glycosyltransferase per gram of the 5, 6-O-isopropylidene-L-ascorbic acid; and/or the dosage of the glucoamylase is as follows: 11500-12000U of glucoamylase is added per gram of the 5, 6-O-isopropylidene-L-ascorbic acid.

3. The method for synthesizing L-ascorbic acid-2-glucoside according to claim 1, wherein an antioxidant is further added during the reaction in step (1), and the amount of the antioxidant is preferably 6-10% of the mass of 5, 6-O-isopropylidene-L-ascorbic acid.

4. The method for synthesizing L-ascorbic acid-2-glucoside of claim 3, wherein the antioxidant is cysteine.

5. The method of synthesizing L-ascorbic acid-2-glucoside of claim 1, wherein the 5, 6-O-isopropylidene-L-ascorbic acid is prepared by reacting L-ascorbic acid with acetone;

the 5, 6-O-isopropylidene-L-ascorbic acid is preferably prepared by the following method that L-ascorbic acid and acetone are fed according to the proportion of L-ascorbic acid to acetone which is 1g (5 ml-10 ml), and any or more of acetyl chloride, phosphorus oxychloride and stannous chloride are adopted as catalysts to catalyze and react to prepare the 5, 6-O-isopropylidene-L-ascorbic acid.

6. The method for synthesizing L-ascorbic acid-2-glucoside of claim 1, wherein said glucose group donor is any of β -cyclodextrin, gelatinized starch, soluble starch, maltodextrin or more .

7. The method for synthesizing L-ascorbic acid-2-glucoside of claim 6, wherein said glycosyltransferase is any or several of α -glucosidase, cyclodextrin glucoside transferase, α -amylase.

8. The method of synthesizing L-ascorbic acid-2-glucoside of claim 1, wherein said glucose-based donor is β -cyclodextrin and said glycosyltransferase is cyclodextrin glucoside transferase.

9. The method for synthesizing L-ascorbic acid-2-glucoside of any of claims 1 or 6 to 8 of , wherein the glucoamylase is α -1, 4-glucohydrolase.

10. The method for synthesizing L-ascorbic acid-2-glucoside according to claim 1, wherein said strongly acidic cation exchange resin is a sulfonic acid group cation exchange resin having a styrene-divinyl copolymer as a matrix.

Technical Field

The invention relates to the technical field of biochemical synthesis, in particular to methods for synthesizing L-ascorbic acid-2-glucoside.

Background

The structural formula of the L-ascorbic acid-2-glucoside (namely 2-O- α -D-glucopyranosyl-L-ascorbic acid) is shown in a formula (I), the L-ascorbic acid-2-glucoside is used as an important derivative of the L-ascorbic acid, is which is a whitening additive published and approved by the health administration, is a commonly recognized stable L-ascorbic acid derivative, is the most commonly used whitening component in various current brand cosmetic products, has good whitening effect, is chemically stable and has no stimulation to skin, so the L-ascorbic acid-2-glucoside is widely used by in cosmetic production at home and abroad, the L-ascorbic acid-2-glucoside entering human body cells is hydrolyzed by α -glucosidase to generate ascorbic acid and glucose, and the L-ascorbic acid-2-glucoside has the same reducing property as ascorbic acid and oxidation resistance, and the L-ascorbic acid-2-glucoside can continuously decompose in vivo to provide the ascorbic acid.

The patent document with application number 201010571709.9 discloses a manufacturing method of anhydrous crystal powder containing 2-O- α -D-glucosyl-L-ascorbic acid, a solution containing starch and L-ascorbic acid is subjected to sequential action of cyclodextrin glucanotransferase and glucoamylase to obtain a solution with 2-O- α -D-glucosyl-L-ascorbic acid productivity of 35% or more, the patent application with application number 201610985353.0 also discloses methods for preparing L-ascorbic acid-2-glucoside, sucrose phosphorylase produced by fermentation of an engineered strain of Escherichia coli using recombinant sucrose phosphorylase as a biocatalyst, sucrose as a glycosyl donor, L-ascorbic acid as a glycosyl acceptor, the preparation of L-ascorbic acid-2-glucoside products by biocatalysis is carried out by using sucrose phosphorylase as a glycosyl acceptor, the reaction of L-ascorbic acid-2-glucoside donor is carried out under the same conditions as L-glucopyranosyl-2-glycosyl transferase, L-ascorbic acid-2-glucopyranoside production reaction of L-ascorbic acid-2-glucose-glycosyl transferase is carried out by using L-ascorbic acid-glucopyranosyl-2-glucopyranoside-glycosyl transferase as a glycosyl donor, L-ascorbic acid donor, L-ascorbic acid is produced by-glucopyranosyl-glucopyranose, L-glucopyranosyl-2-glucopyranoside, L-glucopyranosyl-2-glucopyranoside is produced, L-glucopyranosyl-glucopyranoside production is produced, L-glucopyranosyl-L-glucopyranoside production is produced, L-2-glucopyranoside production is produced, L-glucopyranosyl-glucopyranose-glucopyranosyl-glucopyranose, L-glucopyranosyl-L-glucopyranosyl-L-glucopyranoside production is produced, L-glucopyranosyl-L-glucopyranoside production is produced, L-glucopyranosyl-L-.

The invention aims to find novel reaction substrates as starting materials for synthesizing L-ascorbic acid-2-glucoside so as to reduce the generation of byproducts.

Disclosure of Invention

The invention mainly solves the technical problem of providing a synthesis method of L-ascorbic acid-2-glucoside, which has simple separation and purification and high purity and yield of L-ascorbic acid-2-glucoside products.

In order to solve the technical problems, the invention adopts technical proposals that methods for synthesizing L-ascorbic acid-2-glucoside comprise the following steps:

(1) taking 5, 6-O-isopropylidene-L-ascorbic acid as a reaction raw material, adding a glucose group donor, and carrying out a biotransformation reaction under the action of glycosyltransferase;

(2) adding glucoamylase into the reaction liquid obtained after the reaction in the step (1) for hydrolysis reaction to prepare 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside;

(3) and (3) separating the reaction liquid containing the 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside obtained in the step (2) by adopting a strong acid cation exchange resin, crystallizing the separation liquid to separate out an L-ascorbic acid-2-glucoside product, and returning the residual feed liquid after the product is separated out to the step (1) for recycling. The reactions in the step (1) and the step (2) are carried out under the conditions of light and oxygen avoidance.

Preferably, the dosage of the glucose-based donor is 50-60% of the mass of the 5, 6-O-isopropylidene-L-ascorbic acid; and/or, the glycosyltransferase is used in an amount of: adding 650-700U glycosyltransferase per gram of the 5, 6-O-isopropylidene-L-ascorbic acid; and/or the dosage of the glucoamylase is as follows: 11500-12000U of glucoamylase is added per gram of the 5, 6-O-isopropylidene-L-ascorbic acid.

Preferably, an antioxidant is also added during the reaction in the step (1), and the using amount of the antioxidant is preferably 6-10% of the mass of the 5, 6-O-isopropylidene-L-ascorbic acid.

, preferably, the antioxidant is cysteine.

preferred embodiments are that the 5, 6-O-isopropylidene-L-ascorbic acid is prepared by reacting L-ascorbic acid with acetone, preferably, the L-ascorbic acid and the acetone are fed according to the proportion of the L-ascorbic acid to the acetone which is 1g (5 ml-10 ml), and the 5, 6-O-isopropylidene-L-ascorbic acid is prepared by catalytic reaction by using any or more of acetyl chloride, phosphorus oxychloride and stannous chloride as catalysts, wherein preferably adopts acetyl chloride, the proportion of the L-ascorbic acid to the acetone which is 1g (6 ml-8 ml) is preferably VC, and the reaction is carried out in a water bath at 20-50 ℃, preferably at 30-40 ℃, for 2-5 h, preferably for 4-5 h.

Preferably, the glucose group donor is any or more of β -cyclodextrin, gelatinized starch, soluble starch and maltodextrin.

Preferably, the glycosyl transferase is any or more of α -glucosidase, cyclodextrin glucoside transferase and α -amylase.

More preferably, the glucose-based donor is β -cyclodextrin.

Preferably, the glucoamylase is α -1, 4-glucohydrolase.

Preferably, the strong-acid cation exchange resin is a sulfonic acid group cation exchange resin with a styrene-divinyl copolymer as a matrix.

When the residual feed liquid after the product is separated out is returned to the step (1) for recycling, because the feed liquid still contains reaction materials, such as glucose-based donor, glycosyl transferase, glucoamylase and 5, 6-O-isopropylidene-L-ascorbic acid raw materials, the addition of the glucose-based donor, glycosyl transferase, glucoamylase and the like can be reduced or even avoided according to the actual composition of the residual feed liquid.

The invention provides a synthesis method of L-ascorbic acid-2-glucoside, which comprises the following steps of firstly reacting 5, 6-O-isopropylidene-L-ascorbic acid to prepare 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside, separating reaction liquid obtained by the reaction by using strong acid cation exchange resin, hydrolyzing the 5, 6-O-isopropylidene-L-ascorbic acid-2-glucoside into L-ascorbic acid-2-glucoside, crystallizing and drying to obtain an L-ascorbic acid-2-glucoside product, and recycling residual liquid after crystallization.

The method adopts 5, 6-O-isopropylidene-L-ascorbic acid as a reaction raw material, and 5-hydroxy and 6-hydroxy of the raw material are protected by forming isopropylidene, so that the reaction activity is reduced, the 2-hydroxy is mainly used for reaction, the generation of 5-O- α -D-glucopyranosyl-L-ascorbic acid and 6-O- α -D-glucopyranosyl-L-ascorbic acid by-products is avoided, the subsequent separation and purification difficulty is reduced, the purity and yield of the L-ascorbic acid-2-glucoside product are improved, and the crystallization yield is also improved.

Detailed Description

The technical solution of the present invention will be explained in detail below.