CN108315372A - A kind of method of enzyme process removing trichloro-cane-6-ethyl ester acetyl group - Google Patents

Abstract

The invention discloses a kind of methods that enzyme process removes 6 ethyl ester acetyl group of Sucralose, belong to biocatalysis technology field.For the method for the present invention in organic solvent system, 6 ethyl ester of enzymatic Sucralose removes acetyl group, and Sucralose is made.The advantages that reaction condition of the present invention is mild, and good reaction selectivity, reaction product is single, high conversion rate, product safety prepares Sucralose for biology method and provides new feasible way.

Description

A method for enzymatically deacetylating sucralose-6-ethyl ester

technical field

The invention relates to a method for enzymatically preparing sucralose, belonging to the technical field of biocatalysis.

Background technique

Sucralose is a new type of functional sweetener, and sucralose is a kind of chlorinated sucrose. Its sweetness is 600-800 times that of sucrose. It has high safety, pure taste, cannot be metabolized by the human body, has zero energy value, and has good solubility and stability. agent, which has been approved for use by more than 30 countries.

The synthesis method of sucralose is mainly a chemical method now, which includes two types of whole group protection method and monoesterification method. The whole group protection method (such as US Patent US4617269) uses the steric hindrance of triphenylmethane to tritylate and acylate the three primary hydroxyl groups (6-, 1'-, 6'-positions) of sucrose The remaining 5 secondary hydroxyl groups are obtained, and sucralose is obtained by deprotection, chlorination, and removal of five acyl groups. The obvious disadvantage of this type of reaction is that the process is cumbersome. The monoesterification method (US 4889928, US5023329, US4950746, CN1453285A) is to use chemical means to shield the 6-position hydroxyl group on the sucrose group in the sucrose molecule to produce a monoester, and then through chlorination and removal of the protective group acetyl to obtain Sucralose.

Regardless of the method used to prepare sucralose, deacetylation is a necessary process step for the final sucralose compound to be obtained. Therefore, optimizing the deacetylation step is an important way to improve the efficiency of the sucralose synthesis process.

In the current prior art, we usually use strong alkali solution to achieve the purpose of deacetylation of sucralose-6-ethyl ester. The method is generally carried out in methanol solution with sodium methoxide or sodium hydroxide as a catalyst. With this process, a large amount of sodium ions need to be introduced into the reaction system, and after the product solution containing sucralose is obtained, the sodium ions need to be removed by resin adsorption. This necessarily entails numerous downstream processing and product purification steps. At the same time, due to its harsh pH conditions and temperature, glycosidic bonds may be broken and chlorine atoms may fall off, resulting in many by-products and low yield.

Contents of the invention

The invention discloses a method for preparing sucralose by an enzymatic method, aiming at the shortcomings of using chemical methods to prepare sucralose in the prior art. Specifically, it is a method for obtaining a sucralose product by removing the acetyl group at the 6-position in the sucralose-6-ethyl ester through an enzymatic reaction.

The preparation of sucralose by enzymatic reaction has mild conditions, high selectivity, high conversion rate and easy purification of the product.

Preferably, the enzymatic reaction takes place in a reaction system formed by an organic solvent.

The present invention uses an organic solvent as a reaction system, which can improve the solubility of reactants on the one hand, and on the other hand, uses an organic solvent as a reaction system, and the enzyme in the enzymatic reaction is suspended in the organic solvent, which is easy to recover after the reaction. At the same time, it is worth noting that the use of organic solvents as the reaction system can significantly improve the stability of related enzymes. And by using the organic solvent reaction system, by controlling the concentration, temperature, time and other process conditions, the specificity and selective regulation of the enzyme in the enzymatic reaction can be realized.

In addition, using an organic solvent as a reaction system can also facilitate solvent removal and recovery, and simplify subsequent steps.

Further, the enzymatic method for preparing sucralose disclosed in the present invention is specifically described as follows: in an organic solvent reaction system, sucralose-6-ethyl ester with a concentration of 10-50 g/L is used as a substrate, and 2~10mg/ml enzyme, under the condition of 20~80℃, react for 6-24 hours to obtain sucralose.

Preferably, stirring is constant during the reaction.

Preferably, the present invention further discloses that the enzyme is selected from Lipase AS Amano (from Aspergillus niger), Pig liver esterase, Lipase sp-435 (Candide antarctica), CAL-B (Lipase B from Candida antarctica), Lipase G Amano ( from Penicilliumcamemberti) any one or several.

At the same time, the present invention further discloses that the organic solvent is selected from any one of acetonitrile, tetrahydrofuran, methanol, ethanol, tert-butanol or several mutually soluble ones.

Here, one organic solvent can be selected as the reaction system, or two or more organic solutions can be selected as the reaction system, and it should be noted that when two or more organic solutions are selected as the reaction system, it should be ensured that The organic solutions are mutually soluble.

In addition, the present invention further discloses the steps of: after the reaction is completed, filter, and the reaction clear liquid is concentrated in vacuum, and the solvent is removed to obtain crude sucralose.

Preferably, the temperature of vacuum concentration is 50-80°C.

The above steps can also be: after the reaction is completed, centrifuge, the reaction clear liquid is concentrated in a vacuum, and the solvent is removed to obtain the crude sucralose.

Wherein the temperature of vacuum concentration is 50-80°C.

Preferably, the enzyme is recovered after filtration or centrifugation. Among them, the solid part is enzyme.

After adopting the technical solution disclosed by the invention, compared with the chemical method, the reaction has the advantages of mild reaction conditions, high reaction selectivity, single reaction product, high conversion rate and the like.

Detailed ways

The claims of the present invention will be further described in detail below in conjunction with specific embodiments.

Unless otherwise specified, the reagents used in the present invention are all conventional commercially available reagent products.

Lipase AS Amano (from Aspergillus niger) and Lipase G Amano (from Penicillium camemberti) were purchased from Japan Amano Enzyme Company. Pig liver esterase, Lipase sp-435 (Candide antarctica), CAL-B (Lipase B from Candida antarctica) were purchased from Aladdin Reagent Company.

The synthesis of embodiment 1 sucralose

Dissolve 5 g of sucralose-6-ethyl ester in 100 ml of acetonitrile, add 0.8 g of Lipase AS Amano (from Aspergillus niger), react at 40°C for 24 hours, add 1 g of activated carbon for decolorization, stir at room temperature for 30 minutes, filter to recover the enzyme, and store at 70°C The filtrate was concentrated under vacuum to remove acetonitrile. To obtain 4.3 g of crude sucralose, add 5 ml of water, heat and stir to dissolve. Cool down to room temperature at a constant speed within 5 hours to obtain sucralose crystals. Filter and dry to obtain 4.0 g of the finished product (purity: 91%, yield: 90%).

Example 2 Synthesis of Sucralose

Dissolve 1 g of sucralose-6-ethyl ester in 100 ml of tert-butanol, add 0.6 g of Pig liver esterase, react at 80°C for 14 hours, add 1 g of activated carbon for decolorization, stir at room temperature for 30 minutes, and recover the enzyme by centrifugation, at 70°C The filtrate was concentrated in vacuo to remove tert-butanol. To obtain 0.92 g of crude sucralose, add 5 ml of water, heat and stir to dissolve. Cool down to room temperature at a constant speed within 3 hours to obtain sucralose crystals. Filter and dry to obtain 0.84g of finished product (purity: 96%, yield: 92%).

The synthesis of embodiment 3 sucralose

Dissolve 4 g of sucralose-6-ethyl ester in 100 ml of methanol, add 0.4 g of Lipase sp-435 (Candideantarctica), react at 60°C for 6 hours, add 1 g of activated carbon for decolorization, stir at room temperature for 40 minutes, filter to recover the enzyme, and keep at 50°C Concentrate in vacuo to remove methanol. 3.8 g of crude sucralose was obtained. Add 5ml of water, heat and stir to dissolve. Cool down to room temperature at a constant speed within 5 hours to obtain sucralose crystals. Filter and dry to obtain 3.6g of finished product (purity: 92%, yield: 90%).

Example 4

Dissolve 3 g of sucralose-6-ethyl ester in 100 ml of ethanol, add 1.0 g of CAL-B (Lipase B from Candidaantarctica), react at 20°C for 10 hours, add 1 g of activated carbon for decolorization, stir at room temperature for 40 minutes, and recover the enzyme by filtration. Concentrate in vacuo at 60°C to remove tert-butanol. 2.81 g of crude sucralose was obtained. Add 5ml of water, heat and stir to dissolve. Cool down to room temperature at a constant speed within 5 hours to obtain sucralose crystals. Filtration and drying yielded 2.73 g of the finished product (93% purity, 91% yield).

Example 5

Dissolve 2 g of sucralose-6-ethyl ester in 100 ml of tetrahydrofuran, add 0.2 g of Lipase G Amano (from Penicillium camemberti), react at 50°C for 24 hours, add 1 g of activated carbon for decolorization, stir at room temperature for 40 minutes, and recover the enzyme by filtration, at 50°C Concentrate in vacuo to remove ethanol. 1.96 g of crude sucralose was obtained. Add 5ml of water, heat and stir to dissolve. Cool down to room temperature at a constant speed within 5 hours to obtain sucralose crystals. Filter and dry to obtain 1.8g of finished product (94% purity, 90% yield).

Claims (10)

1. A method for enzymatically removing the acetyl group of sucralose-6-ethyl ester, characterized in that: the method refers to removing the acetyl group at position 6 in sucralose-6-ethyl ester by enzymatic reaction Thereby a method for obtaining a sucralose product. 2. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 1, characterized in that: the enzymatic reaction occurs in a reaction system formed by an organic solvent. 3. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 1 or 2, characterized in that: in an organic solvent reaction system, the three The sucralose-6-ethyl ester is used as the substrate, the enzyme with a concentration of 2-10 mg/ml is added thereto, and the reaction is carried out at 20-80° C. for 6-24 hours to obtain sucralose. 4. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 3, characterized in that: the concentration of the substrate sucralose-6-ethyl ester is 10-50 g/L. 5. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 3, characterized in that: the enzyme is selected from Lipase AS Amano, Pig liver esterase, Lipase sp-435 Any one or more of , CAL-B, Lipase GAmano. 6. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 3, characterized in that: the organic solvent is selected from any of acetonitrile, tetrahydrofuran, methanol, ethanol, and tert-butanol One or several mutually soluble. 7. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 3, characterized in that: it also includes the step of filtering after the reaction, and concentrating the reaction clear liquid in a vacuum to remove solvent to obtain crude sucralose. 8. A kind of enzymatic method for removing the acetyl group of sucralose-6-ethyl ester according to claim 3 is characterized in that: after the reaction finishes, centrifuge, and the reaction clear liquid is vacuum-concentrated, and the solvent is removed to obtain three Crude sucralose. 9. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 7 or 8, characterized in that the temperature of vacuum concentration is 50-80°C. 10. A method for enzymatically deacetylating sucralose-6-ethyl ester according to claim 7 or 8, characterized in that the enzyme is recovered after filtration or centrifugation.