阅读说明：本技术 一种酶法生产抗坏血酸棕榈酸酯的方法 (Method for producing ascorbyl palmitate by enzyme method ) 是由 韩忠睦 曾启明 梁冰 于 2020-04-24 设计创作，主要内容包括：本发明公开了一种酶法生产抗坏血酸棕榈酸酯的方法,包括向生物反应容器中加入棕榈酸甲酯、L-抗坏血酸、吸附剂和固定化脂肪酶；控制反应体系的温度为40℃～45℃,经酶催化转酯化反应获得L-抗坏血酸棕榈酸酯；其中,所述固定化脂肪酶为疏水无纺布复合膜固定化皱褶假丝酵母脂肪酶；且将氨基功能化介孔硅分子筛加作为吸附剂和脂肪酶的辅助固定化载体。本发明制备方法制备的L-抗坏血酸棕榈酸酯在进行较短的酯化反应时间,且在固定化脂肪酶的添加量极少时,所得L-抗坏血酸棕榈酸酯转化率仍然很高；所采用的NH2-MSU-H分子筛吸附剂和疏水无纺布复合膜固定化褶皱假丝酵母脂肪酶使固定化酶的活性较高,有效催化合成L-抗坏血酸棕榈酸酯。(The invention discloses a method for producing ascorbyl palmitate by an enzyme method, which comprises the steps of adding methyl palmitate, L-ascorbic acid, an adsorbent and immobilized lipase into a biological reaction container; controlling the temperature of the reaction system to be 40-45 ℃, and obtaining L-ascorbyl palmitate through enzyme catalysis transesterification; wherein the immobilized lipase is a hydrophobic non-woven fabric composite membrane immobilized candida rugosa lipase; and the amino functionalized mesoporous silicon molecular sieve is added as an adsorbent and an auxiliary immobilized carrier of lipase. The L-ascorbyl palmitate prepared by the preparation method has shorter esterification reaction time, and the conversion rate of the obtained L-ascorbyl palmitate is still high when the addition amount of the immobilized lipase is very small; the adopted NH2-MSU-H molecular sieve adsorbent and hydrophobic non-woven fabric composite membrane are used for immobilizing candida rugosa lipase, so that the immobilized enzyme has higher activity, and L-ascorbyl palmitate is effectively synthesized through catalysis.)

1. An enzymatic method for producing ascorbyl palmitate, which comprises the following steps:

adding methyl palmitate, L-ascorbic acid, an adsorbent and immobilized lipase into a biological reaction vessel; wherein the lipase component of the immobilized lipase is candida rugosa lipase; the carrier of the immobilized lipase is a hydrophobic non-woven fabric composite membrane;

controlling the temperature of the reaction system to be 40-45 ℃, and obtaining the L-ascorbyl palmitate through enzyme catalysis transesterification.

2. The method for producing ascorbyl palmitate by the enzymatic method according to claim 1, wherein the immobilization process of the immobilized lipase comprises:

taking phosphoric acid buffer solution as a solvent, adding Candida rugosa lipase to prepare a solution of 15-20mg/mL, and stirring at room temperature for 30min to prepare a lipase solution;

shearing a hydrophobic non-woven composite membrane with a certain area, cleaning with a phosphoric acid buffer solution, draining, putting the drained composite membrane into a filtering container, and preparing immobilized lipase by adopting a combined immobilization mode of filtering, adsorbing and crosslinking;

the prepared immobilized lipase is dried at the temperature of 30 ℃.

3. The method for producing ascorbyl palmitate in an enzymatic manner according to claim 2, wherein the phosphate buffer solution is a phosphate buffer solution with a pH of 7.4 and a concentration of 25 mM.

4. The method for producing ascorbyl palmitate by the enzyme method according to claim 1, wherein the molar ratio of the methyl palmitate to the L-ascorbic acid is 1: 1-1: 8.

5. The method for producing ascorbyl palmitate by the enzyme method according to claim 1, wherein the addition amount of the adsorbent is 18-22% of the addition mass of the methyl palmitate.

6. The method for producing ascorbyl palmitate by an enzymatic method according to claim 1, characterized in that the adsorbent is an amino-functionalized mesoporous silicon molecular sieve.

7. The method for producing ascorbyl palmitate by the enzyme method according to claim 1, wherein the addition amount of the immobilized lipase is 1-7% of the addition mass of methyl palmitate.

8. The enzymatic ascorbyl palmitate production method according to claim 1, characterized in that the bioreactor vessel is one of a shaker reactor, a rotating packed bed reactor, a vacuum stirred reactor or a fixed bed reactor.

Technical Field

The invention belongs to the technical field of palmitate synthesis, and particularly relates to a method for producing ascorbyl palmitate by an enzyme method.

Background

L-ascorbyl palmitate is a fat-soluble antioxidant, has good nutrition and health care functions of free radical removal, oxidation resistance, aging resistance, cancer resistance, tumor resistance and the like, and is one of the products which are well received in the health care product market at home and abroad. L-ascorbyl palmitate sold in the market at present is basically produced by a chemical catalytic synthesis method, and although the traditional chemical method has mature technology and higher conversion rate, compared with a biological enzyme catalytic synthesis method, the traditional chemical method has the defects of large environmental pollution, more side reactions, high requirement on equipment corrosion resistance and the like.

The reaction condition of the biological enzyme catalysis method is mild, the energy consumption is low, and the method conforms to the policies of energy conservation, emission reduction and low-carbon economy advocated by national economy development. However, there are some problems in the synthesis reaction of the enzyme-catalyzed L-ascorbyl palmitate, such as small experimental scale, poor operation stability of enzyme preparations, and high price of commercial immobilized enzymes, which hinder the large-scale industrial application of the enzyme-catalyzed reaction to a certain extent.

Disclosure of Invention

To overcome the problems in the related art, the present invention provides a method for producing ascorbyl palmitate by an enzymatic method, comprising:

adding methyl palmitate, L-ascorbic acid, an adsorbent and immobilized lipase into a biological reaction vessel; wherein the lipase component of the immobilized lipase is candida rugosa lipase; the carrier of the immobilized lipase is a hydrophobic non-woven fabric composite membrane;

controlling the temperature of the reaction system to be 40-45 ℃, and obtaining the L-ascorbyl palmitate through enzyme catalysis transesterification.

Preferably, the immobilization process of the immobilized lipase comprises:

taking phosphoric acid buffer solution as a solvent, adding Candida rugosa lipase to prepare a solution of 15-20mg/mL, and stirring at room temperature for 30min to prepare a lipase solution;

shearing a hydrophobic non-woven composite membrane with a certain area, cleaning with a phosphoric acid buffer solution, draining, putting the drained composite membrane into a filtering container, and preparing immobilized lipase by adopting a combined immobilization mode of filtering, adsorbing and crosslinking;

the prepared immobilized lipase is dried at the temperature of 30 ℃.

Preferably, the phosphate buffer solution is a phosphate buffer solution with a pH of 7.4 and 25 mM.

Preferably, the molar ratio of the methyl palmitate to the L-ascorbic acid is 1: 1-1: 8.

Preferably, the addition amount of the adsorbent is 18 to 22% of the addition mass of the methyl palmitate.

Preferably, the adsorbent is an amino functionalized mesoporous silicon molecular sieve.

Preferably, the addition amount of the immobilized lipase is 1 to 7% of the addition mass of the methyl palmitate.

Preferably, the bioreactor vessel is one of a shaker reactor, a rotating packed bed reactor, a vacuum stirred reactor or a fixed bed reactor.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention utilizes the methyl palmitate as a reaction substrate, and the reaction system has no other organic solvent except the substrate, replaces the traditional acid and alcohol as raw materials to synthesize the L-ascorbyl palmitate, so that the reaction is rapid, and the continuous high-efficiency production can be realized₂-MSU-H molecular sieve adsorbent, NH used in the invention₂The immobilized candida rugosa lipase of the-MSU-H molecular sieve adsorbent and the hydrophobic non-woven fabric composite membrane can be well adapted, so that the immobilized enzyme has high activity and good thermal stability, and L-ascorbyl palmitate can be effectively catalytically synthesized.

The L-ascorbyl palmitate prepared by the preparation method disclosed by the invention has shorter esterification reaction time, and the conversion rate of the obtained L-ascorbyl palmitate can reach 99.73% when the addition amount of the immobilized lipase is only 7% of that of a substrate methyl palmitate.

After the immobilized lipase provided by the invention participates in the synthesis reaction of L-ascorbyl palmitate for four times, the esterification rate of the L-ascorbyl palmitate can still be maintained at 64.53%, which shows that the stability of the immobilized lipase is very high.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments.

Thus, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The existing enzymatic method for catalytically synthesizing L-ascorbyl palmitate generally directly takes acid and alcohol as raw materials, ester is synthesized by catalysis of lipase, the whole reaction process is slow, continuous and efficient production cannot be realized, enzyme selection is not appropriate, so that the catalytic stability and catalytic activity of the L-ascorbyl palmitate cannot meet ideal requirements, and the conversion rate of the product L-ascorbyl palmitate is not high.

In order to solve the problems, the invention provides a method for replacing a reaction substrate by cheap and easily-obtained methyl palmitate, a reaction system has no other organic solvent except the substrate, so that the reaction is rapid, and continuous and efficient production can be realized.

The Candida rugosa lipase is cheap and easy to obtain compared with other lipases, and is widely applied to food production, but the application of the Candida rugosa lipase in ester synthesis is limited due to the problems of difficult separation and recovery, low enzyme utilization and recovery rate and the like of free Candida rugosa lipase in use, and the immobilized Candida rugosa lipase can improve the utilization rate of the enzyme and is beneficial to continuous production of the enzyme;

therefore, the invention adopts a composite immobilization method of adsorption-filtration-crosslinking by using a hydrophobic non-woven composite membrane as a carrier to prepare the immobilized candida lipase as a transesterification catalyst.

Since the enzyme-catalyzed vinegar synthesis reaction in the organic phase is a thermodynamically controlled reaction, water is a by-product of the reaction. Proper amount of water can improve the activity of enzyme and promote the reaction, but when the water is excessive, the water is an inhibitor which competes with substrate for phthalyl enzyme intermediate, reduces the equilibrium conversion rate and promotes the reverse reaction-hydrolysis reaction, therefore, the invention introduces the adsorbent to carry out dehydration treatment and inhibits the generation of byproduct water;

the inventor finds that compared with the traditional magnesium sulfate, aluminum sulfate and 3A/4A molecular sieve dehydrating agent, after amino is introduced into the pore channel of the pure silicon mesoporous molecular sieve, the original structure of the mesoporous molecular sieve can be kept, meanwhile, the hydrophobicity of the interface of the mesoporous pore channel is improved, the hydrothermal stability of the mesoporous molecular sieve is improved, and the defects of poor thermal stability and low reaction activity of the unmodified mesoporous molecular sieve are effectively overcome;

the organic functionalization method of mesoporous silicon mainly comprises a post-grafting method and a copolycondensation method, wherein the copolycondensation method can anchor organic functional groups on the walls of mesoporous materials and ensure that the distribution of the organic functional groups is relatively uniform, so that the invention adopts the copolycondensation method to anchor amino groups on mesoporous molecular sieves MSU-H to form modified NH₂-a MSU-H molecular sieve.

Further, modified NH in the present invention₂The MSU-H molecular sieve can also play a role of an immobilized auxiliary carrier of lipase in a reaction system, and when the molecular size of the enzyme is matched with the pore diameter of the carrier, the activity of the immobilized enzyme is higher, and the thermal stability is better; when the size of the enzyme molecules is larger than the pore diameter of the carrier, the enzyme molecules are not easy to enter the pore channels of the carrier, the adsorption is easy to occur on the outer surface of the carrier, the activity of the enzyme is low, and the adsorbed enzyme is easy to fall off in the operation process; NH used in the invention₂the-MSU-H molecular sieve and the Candida rugosa lipase can be well adapted, so that the immobilized enzyme is aliveThe performance is further improved, and the thermal stability is better.

The technical solution of the present invention will be explained with reference to specific embodiments.