1. A method for synthesizing acetylhydroxyproline catalyzed by a biological enzyme comprises the step of reacting hydroxyproline and acetate serving as raw materials in an aqueous phase buffer solution containing acyltransferase and having a pH value of 7.0-10.0 to prepare acetylhydroxyproline.

2. The method for synthesizing acetylhydroxyproline catalyzed by biological enzyme as claimed in claim 1, wherein the acetate is selected from ethyl acetate, vinyl acetate, propyl acetate, butyl acetate, etc.

3. The method of claim 1, wherein the acyltransferase is selected from the group consisting of MsACT reported in the literature (Adv. Synth. Catal. volume 360, Issue 24, December 21,2018, Pages 4814-.

4. The method for synthesizing acetylhydroxyproline catalyzed by biological enzyme according to claim 1, wherein in the initial reaction system, the acyltransferase: hydroxyproline: the mass ratio of the acetate is 0.02-0.1:1: 1-2.

5. The method of claim 1, wherein the buffer solution is Phosphate Buffered Saline (PBS)

6. The method for synthesizing acetylhydroxyproline catalyzed by biological enzyme according to claim 1, is characterized by comprising the following steps: sequentially adding the buffer solution, hydroxyproline, acetate and acyltransferase into a reaction container, uniformly stirring, detecting the reaction process by HPLC (high performance liquid chromatography) at the temperature of 25-45 ℃ and under the condition of nitrogen purging, adjusting the pH of a reaction system to 3-5 when the conversion rate reaches 90-99%, filtering by using kieselguhr, adding dichloromethane into filtrate for multiple extraction, and removing the solvent by rotary evaporation to obtain the acetylhydroxyproline product.

Background

Acetyl hydroxyproline with chemical name of N-acetyl-L-4-hydroxyproline and molecular formula of C₇H₁₁NO₄CAS registry number 33996-33-7, having the following structural formula:

collagen contains about 10% hydroxyproline, acetylhydroxyproline is an acetylated modified amino acid derivative, and is a cosmetic/quasi-drug material with barrier protection effect. The acetylhydroxyproline has the effects of keeping moisture for a long time, promoting ceramide synthesis, relieving pruritus of atopic dermatitis patients, repairing fine lines of skin, promoting collagen synthesis, enhancing skin permeability and the like. In the cosmetic industry, the product is mainly used for resisting aging, resisting wrinkle and keeping moisture.

For the synthesis of acetylhydroxyproline, the currently disclosed literature basically adopts a chemical synthesis method, and the invention provides a method for synthesizing acetylhydroxyproline catalyzed by a biological enzyme. The method is characterized in that the acyl transferase is used for catalyzing hydroxyproline to perform ester exchange reaction so as to synthesize the N-acetyl-L-4-hydroxyproline. The method is simple and convenient to operate, the reagent is cheap and easy to obtain, and the method has a good industrial application prospect.

Disclosure of Invention

The invention aims to provide a method for synthesizing N-acetyl-L-4-hydroxyproline by one-step reaction by using hydroxyproline and acetate as raw materials and acyltransferase as a biological enzyme catalyst.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for synthesizing acetylhydroxyproline catalyzed by a biological enzyme comprises the step of reacting hydroxyproline and acetate serving as raw materials in an aqueous phase buffer solution containing acyltransferase and having a pH value of 7.0-10.0 to prepare acetylhydroxyproline.

Preferably, the acyltransferase is selected from MsACT reported in the literature (Adv. Synth. Catal. volume 360, Issue 24, December 21,2018, Pages 4814-.

Preferably, the acetate is selected from ethyl acetate, vinyl acetate, propyl acetate, butyl acetate, and the like.

Preferably, in the initial reaction system, the acyltransferase: hydroxyproline: the mass ratio of the acetate is 0.02-0.1:1: 1-2.

Preferably, the buffer solution is Phosphate Buffered Saline (PBS).

Preferably, the specific implementation process is as follows: sequentially adding the buffer solution, hydroxyproline, acetate and acyltransferase into a reaction container, uniformly stirring, detecting the reaction process by HPLC (high performance liquid chromatography) at the temperature of 25-45 ℃ and under the condition of nitrogen purging, adjusting the pH of a reaction system to 3-5 when the conversion rate reaches 90-99%, filtering by using kieselguhr, adding dichloromethane into filtrate for multiple extraction, and removing the solvent by rotary evaporation to obtain the acetylhydroxyproline product.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: according to the invention, acetyl hydroxyproline is synthesized by catalyzing the ester exchange reaction of hydroxyproline and acetate by acyltransferase, the substrate stability is good, the enzyme conversion rate is high, the operation is simple and convenient, no protein residue is generated in subsequent products, and the product purity is high in the whole preparation process.

Detailed Description

The reaction formula of the invention is as follows:

the present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.

Example 1

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 0.5 g of acyltransferase MsACT, 10 g of hydroxyproline and 11 g of ethyl acetate are sequentially added, and the mixture is reacted for 24h under the conditions of 25 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 12 g of product with 98% purity.

Example 2

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.5, 0.5 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of vinyl acetate are sequentially added, and the mixture is reacted for 24h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 11 g of product with 98% purity.

Example 3

In a 500mL three-necked flask, 200mL of 0.1M pH 9PBS buffer solution, 0.4 g of acyltransferase MsACT, 10 g of hydroxyproline and 12 g of propyl acetate are sequentially added, and the mixture is reacted for 24h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging at 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 10 g of product with 98% purity.

Example 4

In a 500mL three-neck flask, 0.1M pH 9PBS buffer solution 200mL, acyltransferase MsACT 0.4 g, hydroxyproline 10 g and butyl acetate 13 g are sequentially added, and the reaction is carried out for 24h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging at 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 9 g of product with 98% purity.

Example 5

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 0.5 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 24h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 12 g of product with 98% purity.

Example 6

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 0.4 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 24h under the conditions of 25 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 11 g of product with 98% purity.

Example 7

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 0.4 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 22h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 12 g of product with 98% purity.

Example 8

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.5, 0.4 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 16h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging by 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 11 g of product with 98% purity.

Example 9

In a 500mL three-necked flask, 200mL of 0.1M PBS buffer solution with pH of 8.5, 0.4 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 13h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging at 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 10 g of product with 98% purity.

Example 10

In a 500mL three-necked flask, 200mL of 0.1M pH 9PBS buffer solution, 0.5 g of acyltransferase MsACT, 10 g of hydroxyproline and 10 g of ethyl acetate are sequentially added, and the mixture is reacted for 10h under the conditions of 30 ℃, stirring by a stirring paddle at 200rpm and nitrogen purging at 0.01MPa, and the conversion rate is 95 percent by HPLC. Hydrochloric acid was added to adjust the pH to 3-5, celite filtered, extracted twice with an equal volume of dichloromethane and rotary evaporated to give 9 g of product with 98% purity.