阅读说明：本技术 一种在高水活度介质中催化合成l-抗坏血酸油酸酯的方法 (Method for catalytically synthesizing L-ascorbyl oleate in high-water-activity medium ) 是由 姚冬生 郑少燕 芦根 刘大岭 谢春芳 刘桂祯 黄炯威 劳伟明 于 2020-07-17 设计创作，主要内容包括：本发明属于生物工程技术领域,涉及一种在高水活度介质中催化合成L-抗坏血酸油酸酯的方法。本发明所述的在高水活度介质中催化合成L-抗坏血酸油酸酯的方法,包括以下步骤：以油酸乙酯和L-抗坏血酸为底物,混合后加入脂肪酶,置于高水活度的水/脂质双相介质中,由重组脂肪酶催化合成L-抗坏血酸油酸酯；所述重组脂肪酶是基于氨基酸序列如SEQ ID NO.1所示的脂肪酶的重组脂肪酶。本发明首次发现CpLIP2脂肪酶可催化油酸乙酯与L-抗坏血酸合成L-抗坏血酸油酸酯,且催化反应可在高水活度(a<Sub>w</Sub>>0.9)水/脂质双相介质中进行。本发明所述方法能在不含有机溶剂的水性介质中通过脂肪酶生产L-抗坏血酸油酸酯,反应条件简单,提供了一种有效的生物转化生产绿色油脂的方法。(The invention belongs to the technical field of bioengineering, and relates to a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium. The method for catalytically synthesizing the L-ascorbic acid oleate in the high-water-activity medium comprises the following steps of: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown as SEQ ID No. 1. The invention discovers for the first time that CpLIP2 lipase can catalyze ethyl oleate and L-ascorbic acid to synthesize L-ascorbic acid oleate, and the catalytic reaction can be carried out at high water activity (a) w >0.9) in a water/lipid biphasic medium. The method of the inventionThe method can produce the L-ascorbyl oleate by lipase in an aqueous medium without organic solvent, has simple reaction conditions, and provides an effective method for producing the green grease by biotransformation.)

1. A method for catalytically synthesizing L-ascorbyl oleate in a medium with high water activity is characterized by comprising the following steps: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown in SEQ ID No.1 and derived from Candidapapesisis.

2. The process for the catalytic synthesis of L-ascorbyl oleate in a high water activity medium according to claim 1, characterized in that: water activity a of the water/lipid biphasic medium_w>0.9。

3. The method of claim 1, wherein: the water/lipid biphasic medium is 50mM phosphate buffer solution with pH6.5 and ethyl oleate emulsion.

4. The method of claim 1, wherein: the process for synthesizing the L-ascorbyl oleate by the catalysis of the recombinant lipase comprises the following steps: stirring the reaction system at 40 ℃ and 180rpm, reacting for 2.5 hours, and separating and purifying from the reaction system to obtain the L-ascorbyl oleate.

5. The method according to claim 4, wherein the separation and purification method comprises the following steps: 2mL of ethyl acetate and 0.792g of sodium chloride are added into each 2.2mL of reaction system, mixed uniformly for 3min by vortex, L-ascorbyl oleate is extracted, and then centrifuged for 5min at 5000 rpm. Taking the supernatant, heating the supernatant to 77 ℃, and volatilizing ethyl acetate to obtain the L-ascorbyl oleate.

6. The method of claim 1, wherein: the concentration of the added reaction substrate L-ascorbic acid in the reaction system is 0.2-2.2mol/L, and the final concentration of the ethyl oleate is 15-250 mmol/L.

7. The method of claim 1, wherein: the recombinant lipase is a recombinant lipase secreted and expressed by yeast.

8. The method of claim 7, wherein: the final concentration of the recombinant lipase in the reaction system is 0.05-2 mg/mL.

9. The method of claim 1, wherein: the recombinant lipase is a recombinant lipase displayed by yeast.

10. The method of claim 9, wherein: the final concentration of the recombinant lipase in the reaction system is 0.01-0.5 g/mL.

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium. More specifically, the invention provides a method for synthesizing L-ascorbic acid oleate by taking ethyl oleate and L-ascorbic acid as raw materials and catalyzing ethyl oleate in a high-water-activity medium through lipase.

Background

The oil is one of important nutrient substances required by human bodies, and the oil in the food is easily oxidized and rancid in the processing, storage and transportation processes, so that the nutritional value of the food is reduced, and even the health of the human body is damaged. The addition of antioxidants to foods is often an effective way to solve this problem. The L-ascorbic acid fatty acid ester is a fat-soluble derivative of the L-ascorbic acid, and compared with the L-ascorbic acid saturated fatty acid ester, the L-ascorbic acid oleate has higher solubility in oil substances and better antioxidant effect, and is more suitable for the antioxidation of the oil.

Lipases are water-soluble enzymes with various catalytic abilities and capable of playing an active role in an oil-water interface, and can catalyze hydrolysis of esters such as fats. Under certain conditions, lipases are also capable of catalyzing the production of esters, the types of reactions being esterification, alcoholysis, acidolysis and transesterification. Since water is a product of the ester synthesis reaction, organic solvents with very low water activity are typically used and water formed during the reaction must be extracted, transesterification reactions (such as alcoholysis) do not produce water, but they should also typically be carried out at low water activity to prevent hydrolysis of the ester. Therefore, the synthesis of L-ascorbyl oleate catalyzed by the enzyme method is basically carried out in a non-aqueous phase system, such as an organic solvent. For example, an invention patent (publication No. CN 101892275B) entitled "method for synthesizing ascorbic acid fatty acid ester by catalyzing lard with lipase" discloses that isooctane: t-amyl alcohol with a volume ratio of 7: 3 is used as a reaction medium, alcoholysis reaction is carried out on lard and methanol with lipase to prepare composite fatty acid methyl ester, and then transesterification reaction is carried out on the composite fatty acid methyl ester and L-ascorbic acid to prepare ascorbic acid fatty acid ester; an invention patent (publication No. CN 102212572B) named as a method for synthesizing L-ascorbyl oleate by catalysis of yeast display lipase discloses that L-ascorbyl oleate is synthesized by catalysis of L-ascorbic acid and oleic acid by taking tetrahydrofuran as a reaction medium. There is a document (https:// doi.org/10.5650/jos.62.591) that L-ascorbic acid oleate is synthesized under catalysis of lipase using acetone as a reaction medium and L-ascorbic acid and oleic acid as substrates.

At present, L-ascorbyl oleate is not commercially produced, and the research on the synthesis of L-ascorbyl oleate is mainly carried out by using lipase in an organic solvent, but the use of the organic solvent not only causes many safety problems, but also increases the downstream processing cost of L-ascorbyl fatty acid ester serving as a green food additive. The synthesis of the organic phase causes environmental pollution, and the residual organic solvent in the sample may be harmful to human bodies. For example, the published patents: CN 102212572B (method for catalyzing and synthesizing L-ascorbyl oleate by using yeast display lipase) and CN 102212574B (method for catalyzing and synthesizing L-ascorbyl linoleate by using yeast display lipase) both use tetrahydrofuran organic solvent as a reaction medium to synthesize L-ascorbyl oleate/linoleate. Although more environmentally friendly, new reaction media such as ionic liquids, supercritical fluids, etc. have high cost and harsh operating conditions, which limit their large-scale application.

Studies have reported that high yields of fatty acid esters of glycerol or methanol are feasible in aqueous/lipid biphasic media. Candida deformans derived lipase (CdIP 1) (https:// doi.org/10.1002/yea.958) was able to esterify to methyl oleate in the presence of methanol and either oleic acid or triolein in aqueous solution. Candidapapsilosis-derived lipase (CpLIP2) catalyzes the alcoholysis of methanol and rapeseed oil in aqueous solution. Rodrigues (https:// doi.org/10.1016/j.biortech.2016.07.090) and the like immobilize the enzyme on two synthetic resins, Accurel MP 1000 and Lewatit VP OC 1600, and catalyze the transesterification of jatropha oil and methanol (2M) to synthesize biodiesel in a lipid/water system (aw-0.96). Although these studies suggest the possibility of direct bioconversion of certain specific lipases in aqueous media to synthesize methyl oleate and biodiesel, no synthesis of L-ascorbyl oleate has been reported with lipases catalyzed by ethyl oleate and L-ascorbic acid in aqueous media.

Therefore, the development of effective bioconversion in organic solvent free aqueous media is particularly challenging for green oleochemistry and also an important development direction for green oleochemistry in the future.

Disclosure of Invention

The invention aims to provide a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium, which can produce the L-ascorbyl oleate by lipase in an aqueous medium without an organic solvent, has simple reaction conditions and provides a new way for green and healthy oil production.

The method for catalytically synthesizing the L-ascorbic acid oleate in the high-water-activity medium comprises the following steps of: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown in SEQ ID No.1 and derived from Candidapapesisis.

According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity, the waterWater Activity of the lipid biphasic Medium a_w>0.9。

According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the water/lipid biphasic medium is a 50mM phosphate buffer solution at pH6.5 and an ethyl oleate emulsion.

According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity of the present invention, the process for the catalytic synthesis of L-ascorbyl oleate by the recombinant lipase is: stirring the reaction system at 40 ℃ and 180rpm, reacting for 2.5 hours, and separating and purifying from the reaction system to obtain the L-ascorbyl oleate.

According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a medium with high water activity of the present invention, the separation and purification method comprises: 2mL of ethyl acetate and 0.792g of sodium chloride are added into each 2.2mL of reaction system, mixed uniformly for 3min by vortex, L-ascorbyl oleate is extracted, and then centrifuged for 5min at 5000 rpm. Taking the supernatant, heating the supernatant to 77 ℃, and volatilizing ethyl acetate to obtain the L-ascorbyl oleate.

According to a further feature of the method for catalytically synthesizing L-ascorbic acid oleate in a medium with high water activity of the present invention, the concentration of the added reaction substrate L-ascorbic acid in the reaction system is 0.2-2.2mol/L, and the final concentration of ethyl oleate is 15-250 mmol/L.

According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the recombinant lipase is a recombinant lipase secreted and expressed by yeast.

According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a high water activity medium, the final concentration of the recombinant lipase in the reaction system is 0.05-2 mg/mL.

According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the recombinant lipase is a recombinant lipase displayed by yeast.

According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a high water activity medium, the final concentration of the recombinant lipase in the reaction system is 0.01-0.5 g/mL.

The invention introduces lipase gene and cell wall α -agglutinin gene into Pichia pastoris GS115, after the Pichia pastoris cell is cultured, the lipase is expressed and secreted to the outside of cell, or the cell wall α -agglutinin is utilized to fix the lipase on the cell surface, the reaction of synthesizing L-ascorbic acid oleate by utilizing the yeast display lipase to take L-ascorbic acid and ethyl oleate as substrates is catalyzed_w>0.9), the lipase used in the invention can also catalyze the synthesis of L-ascorbic acid oleate from L-ascorbic acid and ethyl oleate in the form of immobilized enzyme in a reaction medium. Therefore, the invention provides an effective biotransformation method for producing green oil.

The invention discovers for the first time that CpLIP2 lipase can catalyze ethyl oleate and L-ascorbic acid to synthesize L-ascorbic acid oleate, and the catalytic reaction can be carried out at high water activity (a)_w>0.9) in a water/lipid biphasic medium, the CpLIP2 lipase displayed by the cells has high operation stability, the conversion rate (calculated by L-ascorbic acid) is more than 70.3 percent, and the yield is more than 65.8 percent.

Drawings

FIG. 1 is a figure showing the mass spectrometric identification of L-ascorbyl oleate synthesized by the lipase catalysis of the invention.

FIG. 2 is a fluorescent microscopic examination result of pPCPA-GS 115.

Detailed Description

The present invention will be further described in detail by the following specific examples in conjunction with the attached drawings, wherein the examples are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation procedures are given, but the scope of the present invention is not limited to these examples.