阅读说明：本技术 一种脂肪酶催化合成维生素a二十二碳六烯酸酯的方法 (Method for synthesizing vitamin A docosahexaenoic acid ester under catalysis of lipase ) 是由 毛相朝 孙建安 高坤鹏 于 2020-06-02 设计创作，主要内容包括：本发明属于酶催化合成技术领域,公开了一种脂肪酶催化合成维生素A二十二碳六烯酸酯的方法。本发明所述方法利用固定化脂肪酶Novozyme435催化乙酯型鱼油与维生素A醋酸酯发生酯交换反应,合成维生素A二十二碳六烯酸酯,所述酯交换反应是将维生素A醋酸酯和乙酯型鱼油溶解于有机溶剂中,加入固定化脂肪酶Novozyme435,于无氧条件下进行反应后分离纯化得到维生素A二十二碳六烯酸酯。该方法利用脂肪酶Novozyme435催化乙酯型鱼油与维生素A醋酸酯发生酯交换反应,首次合成了维生素A二十二碳六烯酸酯,制备的维生素A二十二碳六烯酸酯稳定性很好,杂质含量低,且反应温度低,能够在较短时间实现反应底物的转化,较好地避免了原料的变质。(The invention belongs to the technical field of enzyme catalytic synthesis, and discloses a method for synthesizing vitamin A docosahexaenoic acid ester through lipase catalysis. The method utilizes immobilized lipase Novozyme435 to catalyze ethyl ester type fish oil to perform ester exchange reaction with vitamin A acetate to synthesize the vitamin A docosahexaenoate, wherein the ester exchange reaction is to dissolve the vitamin A acetate and the ethyl ester type fish oil in an organic solvent, add the immobilized lipase Novozyme435, perform reaction under anaerobic condition, and then separate and purify to obtain the vitamin A docosahexaenoate. According to the method, lipase Novozyme435 is used for catalyzing ethyl ester type fish oil to perform an ester exchange reaction with vitamin A acetate, so that vitamin A docosahexaenoate is synthesized for the first time, the prepared vitamin A docosahexaenoate has good stability, low impurity content and low reaction temperature, the conversion of a reaction substrate can be realized in a short time, and the deterioration of raw materials is well avoided.)

1. A method for synthesizing vitamin A docosahexaenoate under catalysis of lipase is characterized in that immobilized lipase Novozyme435 is used for catalyzing ethyl ester type fish oil to perform transesterification with vitamin A acetate, and the vitamin A docosahexaenoate with the following structure is synthesized:

2. the method for synthesizing vitamin A docosahexaenoic acid ester through lipase catalysis as claimed in claim 1, wherein the transesterification reaction is to dissolve vitamin A acetate and ethyl ester type fish oil in an organic solvent, add immobilized lipase Novozyme435, perform reaction under anaerobic condition, and then separate and purify to obtain vitamin A docosahexaenoic acid ester.

3. The method for the catalytic synthesis of vitamin A docosahexaenoic acid ester according to claim 1, wherein the volume ratio of the organic solvent to the ethyl ester type fish oil is 80-120: 1.

4. the method for the catalytic synthesis of vitamin A docosahexaenoic acid ester according to claim 1, wherein 0.0025-0.0075 mM vitamin A acetate is added into each μ L fish oil.

5. The method for the catalytic synthesis of vitamin A docosahexaenoic acid ester according to claim 1, wherein 200-300 mg of immobilized lipase is added to each mM of vitamin A acetate.

6. The method for the lipase-catalyzed synthesis of vitamin A docosahexaenoic acid ester according to claim 1, wherein the organic solvent is a lipophilic organic solvent.

7. The method for the lipase-catalyzed synthesis of vitamin A docosahexaenoic acid ester according to claim 6, wherein the organic solvent is one or more of petroleum ether, n-hexane, cyclohexane and isooctane.

8. The method for the lipase-catalyzed synthesis of vitamin A docosahexaenoic acid ester as claimed in claim 7, wherein the organic solvent is petroleum ether.

9. The method for the catalytic synthesis of vitamin A docosahexaenoic acid ester according to claim 1, wherein the reaction temperature is 25-40 ℃ and the reaction time is 1-24 h.

10. The method for synthesizing vitamin A docosahexaenoic acid ester under the catalysis of lipase according to claim 1, wherein the separation and purification method comprises the following steps: centrifuging to remove enzyme, removing organic solvent from supernatant, and purifying by C18 column chromatography.

Technical Field

The invention relates to the technical field of enzyme catalytic synthesis, in particular to a method for synthesizing vitamin A docosahexaenoic acid ester under the catalysis of lipase.

Background

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid widely present in marine fish oil, and has various effects of preventing and treating cardiovascular diseases, treating diabetes, inhibiting tumor cells, etc. In addition, DHA is an important component of retina, and plays an important role in the development of vision. At present, DHA with high content and capable of being stably existing in total fatty acid is mainly in an ethyl ester form, and the ethyl ester form is not beneficial to human body absorption and has low bioavailability. Some existing researches try to modify Lipase (Lipase, EC 3.1.1.3) by utilizing the specific catalytic activity of the Lipase to synthesize glyceride and phospholipid lipid forms with higher DHA content, so that the high DHA content in the total fatty acids is retained, and the DHA is converted into a more easily absorbed form.

Vitamin A is one of essential vitamins for human bodies, is an essential vitamin in the growth process of children and is the earliest discovered vitamin, and has important effects on maintaining normal visual response and treating nyctalopia, xerophthalmia and the like. However, vitamin a is extremely unstable, easily oxidatively decomposes in light, air, and high temperature, and is irritating to the skin. In order to reduce its instability and irritation, various methods have been used to convert vitamin a into vitamin a esters, and vitamin a esters widely used in the market mainly include vitamin a palmitate, vitamin a acetate, vitamin a oleate, vitamin a succinate, and the like.

Disclosure of Invention

The invention aims to combine the functions of promoting the development of vision and protecting vision of DHA, and to use vitamin A to carry out esterification modification on DHA, so as to expect to obtain a compound which is more beneficial to the development of vision and keeps the health of vision.

In order to achieve the purpose of the invention, the immobilized lipase Novozyme435 is used for catalyzing the ethyl ester type fish oil to perform ester exchange reaction with vitamin A acetate, and vitamin A docosahexaenoate with the following structure is synthesized:

further, the ester exchange reaction is to dissolve vitamin A acetate and ethyl ester type fish oil in an organic solvent, add immobilized lipase Novozyme435, perform reaction under an anaerobic condition, and then separate and purify to obtain the vitamin A docosahexaenoate.

Further, the volume ratio of the organic solvent to the ethyl ester type fish oil is 80-120: 1.

furthermore, 0.0025-0.0075 mM of vitamin A acetate is added into each mu L of fish oil.

Further, 200-300 mg of immobilized lipase is added into each mM of vitamin A acetate.

Further, the organic solvent is a lipophilic organic solvent.

Preferably, the organic solvent is one or more of petroleum ether, n-hexane, cyclohexane and isooctane.

More preferably, the organic solvent is petroleum ether.

Further, the reaction temperature is 25-40 ℃, and the reaction time is 1-24 h.

Further, the separation and purification method comprises the following steps: centrifuging to remove enzyme, removing organic solvent from supernatant, and purifying by C18 column chromatography.

According to the invention, lipase Novozyme435 is used for catalyzing ethyl ester type fish oil to perform an ester exchange reaction with vitamin A acetate, so that vitamin A docosahexaenoate is synthesized for the first time, a foundation is laid for the development of related researches on the physiological activity of the compound, more references are provided for the research field, and more selectivity is provided for industrial application. In addition, the prepared vitamin A docosahexaenoate has good stability, low impurity content and low reaction temperature, can realize the conversion of reaction substrates in a short time, and better avoids the deterioration of raw materials.

Drawings

FIG. 1 shows the results of detection and identification of a target product in example 1 of the present invention;

FIG. 2 is the results of examining the yields of products obtained by using different organic solvents in example 3 of the present invention;

FIG. 3 is a graph showing the effect of the amount of vitamin A acetate added on the yield of the resulting product in example 4 of the present invention;

FIG. 4 is a graph showing the results of examining the yields of products obtained at different reaction temperatures in example 5 according to the present invention;

FIG. 5 is a graph showing the effect of enzyme addition on the yield of the resulting product in example 6 of the present invention;

FIG. 6 is a graph showing the change of the efficiency of the transesterification reaction with time in example 7 of the present invention;

FIG. 7 is a graph showing the effect of enzyme use batches on the efficiency of catalytic product formation in example 8 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be construed as limiting the present invention.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.