Method for preparing phosphatidylserine through interfacial enzyme catalysis

Abstract

The invention belongs to the field of enzymatic preparation of structural phospholipids, and particularly relates to a method for preparing phosphatidylserine through interfacial enzyme catalysis. A series of high-activity immobilized phospholipase are prepared by innovatively adopting bifunctional group modified hollow mesoporous silica microspheres as a carrier and adsorbing immobilized phospholipase D. The catalyst has the functions of a catalyst and an emulsifier, and a Pickering emulsion interfacial enzyme catalysis system can be efficiently constructed. In the system, the phosphatidylcholine and the serine are efficiently converted into the phosphatidylserine through a phosphatidyl transfer reaction, the conversion rate of the phosphatidylserine within 20 minutes can reach 85%-95%, and the catalytic efficiency reaches up to 372 mmol / (g.h). In addition, after the catalyst is recycled for 10 times, the conversion rate is still kept at 70.9%, and the catalyst shows excellent stability and reusability. The method has the remarkable advantages of being mild in reaction condition, high in catalytic efficiency, capable of being repeatedly used and the like, and a new technical approach is provided for industrial production of phosphatidylserine.

Description

Technical Field

[0001] The present invention belongs to the technical field of structural phospholipid synthesis, and particularly relates to a method for preparing phosphatidylserine by interfacial enzyme catalysis. Background Art

[0002] Phosphatidylserine (PS) is a phospholipid with important biological functions and application values. The traditional method for preparing PS mainly involves the phosphatidyl group transfer reaction of phosphatidylcholine (PC) and serine (L-Ser) catalyzed by phospholipase D (PLD). This method has mild reaction conditions and high specificity, but is limited by the small interfacial area of the traditional organic-aqueous two-phase enzyme catalysis system and the poor stability of free enzymes, resulting in low enzymatic catalysis efficiency. For example, in the method described in Patent CN103131737A, after 4 hours of reaction, the PS yield is only 40%. In addition, although the yield of PS prepared using a pure water system is as high as 99.5%, due to the low solubility of the substrate in water, it is difficult to meet the requirements of industrial production. Summary of the Invention

[0003] In view of the above deficiencies in the prior art, the present invention aims to provide a method for preparing PS through an interfacial enzyme catalysis system, using a mesoporous support modified with bifunctional groups to immobilize lipase, stabilizing the interfacial enzyme catalysis system, and efficiently preparing phosphatidylserine.

[0004] It should be noted that in response to the above technical bottleneck, the present invention proposes a new method for efficiently preparing PS based on a Pickering interfacial enzyme catalysis system stabilized by immobilized enzyme particles. By introducing nitrogen-containing alkylamine groups and n-octyl groups on the surface of hollow mesoporous silica-based particles (HMSPs), the synergistic optimization of hydrogen bond interaction and interfacial activation effect between the support and PLD is realized, significantly enhancing the stability and catalytic activity of the immobilized enzyme particles. The bifunctional modified support, interfacial enzyme catalysis system, and process method constructed by the present invention have opened up a new technical paradigm for the green biofabrication of structural phospholipid compounds.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preparing phosphatidylserine by interfacial enzyme catalysis, comprising the following steps:

[0007] (1) Preparation of immobilized enzyme: Free phospholipase D was added to phosphate buffer to obtain an enzyme solution diluted twofold. Then, the enzyme solution was mixed with a carrier (HMSP-N3 / C8) modified with NQ-62 and OTCS, sonicated for 5 minutes, filtered by suction for 5 minutes, shaken in a shaker at 30 °C for 2 hours, and then freeze-dried to obtain immobilized phospholipase (PLD@HMSP-N3 / C8). Experiments showed that the SiO2 carrier was a nanoscale hollow mesoporous SiO2 nanosphere modified with amino groups and silane coupling agents, with a size of about 500 nm, a mesopore size distribution between 10 - 20 nm, and a water contact angle of 45° - 110°, which was beneficial to the mass transfer and heat transfer of reaction substrates in the system and the efficient immobilization of enzymes. Moreover, the introduction of nitrogen-containing alkyl amino groups increased the hydrogen bond interaction between the enzyme and the carrier, improving its stability. The introduction of n-octyl provided an appropriate hydrophobic microenvironment for phospholipase, stimulating its "interface activation effect" and improving enzyme activity.

[0008] (2) Preparation of Pickering emulsion interfacial enzyme catalytic system: Phosphatidylcholine was dissolved in ethyl butyrate, and serine was dissolved in PBS (pH = 6 mM). They were mixed in a ratio of 4:6, and the immobilized phospholipase D (PLD@HMSP-N3 / C8) obtained in step (1) was added. Then, high-pressure homogenization or vigorous stirring was carried out for 1 minute to form a nanoscale emulsion, and then the transphosphatidylation reaction was carried out. Samples were taken at regular intervals for detection.

[0009] (3) After the reaction in step (2) was completed, the immobilized phospholipase was separated and reused, and the obtained solution was dried by blowing with nitrogen to obtain phosphatidylserine.

[0010] Preferably, in step (1), the free enzyme source belongs to phospholipase D of the genus Bacillus, the protein concentration of the phospholipase D enzyme solution is 4.6 mg / mL, the pH of the enzyme solution is 4 - 8; the ratio of the mass of the carrier to the volume of the enzyme solution is 100:10 - 500:10 (m / v, mg / mL); the immobilization time is 30 - 150 min, and the temperature is 20 - 40 °C; the pH of the phosphate buffer is 4 - 8, and the immobilization amount is 60 - 90 mg / g.

[0011] Preferably, in step (2), the concentration of phosphatidylcholine dissolved in ethyl butyrate is 10 - 50 mg / mL, the concentration of serine dissolved in PBS is 1 - 4 M, the temperature of the transphosphatidylation reaction is 30 - 70 °C, the reaction time is 20 - 60 min, the pH is 4 - 8, and the addition amount of the immobilized enzyme is 0.5 - 2.0 wt% of the reaction system. A stable water-in-oil Pickering emulsion system with an average emulsion droplet diameter of 80 - 88.9 μm and a specific surface area of the emulsion reaching 582.2 - 669.6 cm 2 。

[0012] Preferably, in step (2), the high-pressure homogenization is carried out by using a high-pressure homogenizer for 10 - 20 cycles, and the pressure is 400 - 600 Pa.

[0013] It should be noted that in step (2), phosphatidylcholines from various sources can be used as substrates, such as those from egg yolk, soybean, animal liver, fish, nut seeds, and dairy products.

[0014] Preferably, the immobilized enzyme separated in step (3) can be reused after being washed with PBS and freeze-dried; when the immobilized enzyme is reused 10 times, the content of phosphatidylserine prepared remains above 70%.

[0015] Preferably, in step (3), the phosphatidylserine obtained by transformation should be separated and purified to obtain a phosphatidylserine product. The specific steps are as follows: The low-temperature centrifugation method (4°C, 5000 rpm, 10 min) is used to induce the aggregation of emulsion droplets to achieve rapid stratification of the oil and water phases; the demulsified aqueous phase (containing PS product) is used to collect phosphatidylserine in a chloroform / methanol volume ratio of 2:1 collecting solution, and extracted three times, and the organic solvent is removed by vacuum distillation (40 - 50°C, 0.1 MPa) to obtain the PS product.

[0016] It should be noted that the method of the present invention can not only prepare this kind of structural phospholipid (phosphatidylserine), but also prepare other structural phospholipids according to the similarity of the serine structure, such as phosphatidylthreonine.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. Aiming at the problem of low efficiency of PS esterification synthesis, based on the interfacial mechanism of enzyme-catalyzed reactions, the present invention constructs a Pickering interfacial biocatalytic system with both emulsification and catalytic functions. This system uses a carrier precisely modified with dual functional groups to immobilize the enzyme directionally, which not only effectively stabilizes the spatial conformation of the enzyme, maintains its high catalytic activity, but also regulates the spatial orientation of the enzyme so that its active center faces the oil-water interface, significantly improving the substrate accessibility and the enzyme-substrate interaction efficiency. In addition, the immobilized enzyme itself has emulsifying properties and can induce the stable formation of Pickering emulsion without the addition of external emulsifiers, thus significantly expanding the reaction area of the oil-water interface, strengthening the interfacial mass transfer process, and synergistically improving the esterification reaction efficiency. The PS conversion rate reaches 94% within 20 min, providing a new way for the synthesis of PS.

[0019] 2. The reaction process of the present invention uses a Pickering emulsion interface enzyme-catalyzed system, with mild reaction conditions and short reaction time; it avoids the environmental pollution problems in chemical catalytic methods, and the reaction efficiency is greatly improved compared with traditional enzyme-catalyzed methods. It is a green, widely applicable, and highly efficient PS enzyme-based directional preparation technology.

[0020] 3. The present invention designs and constructs a novel mesoporous silica-based support modified with dual functional groups for the efficient immobilization of phospholipase. Through reasonable screening of the modifying groups, an alkyl chain and a nitrogen-containing functional group are introduced. By regulating the number and distribution of nitrogen atoms in its structure, the hydrogen bond and electrostatic interaction between the support and the enzyme molecule are significantly enhanced, thereby improving the binding efficiency and conformational stability of the enzyme during the immobilization process, enhancing the operational reusability and catalytic tolerance of the immobilized enzyme, extending its service life, and reducing the unit enzyme activity cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative efforts.

[0022] Figure 1 It is a schematic diagram of a method for preparing phosphatidylserine by interfacial enzyme catalysis.

[0023] Figure 2 They are the TEM and contact angle of hollow mesoporous SiO2 nanospheres modified with amino and silane coupling agents.

[0024] Figure 3 It is a fluorescence confocal microscopy image of the emulsion obtained before the reaction in Example 1, scale bar: 60 μm.

[0025] Figure 4 It is the reusability of the Pickering system in the PS conversion in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be interpreted as superior or better than other embodiments. For the performance index tests in the embodiments of the present application, unless otherwise specified, the conventional test methods in the art are adopted. It should be understood that the terms described in the present application are only for describing specific embodiments and are not used to limit the content disclosed in the present application.

[0028] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which this application belongs; other test methods and technical means not specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0029] In order to better illustrate the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that this application can still be implemented without certain specific details. In the embodiments, some methods, means, instruments, devices, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of this application.

[0030] On the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of this application.

[0031] The present invention discloses a method for preparing phosphatidylserine by interfacial enzyme catalysis.

[0032] In the present invention, the specific preparation process of SiO2 nanospheres is as follows: First, CTAC (25 g) is dissolved in water (100 mL) to form a 25 wt% aqueous solution; 0.83 mL of the CTAC aqueous solution is taken, and ethanol (8 mL) and ammonia water solution (25%, 0.2 mL) are added in sequence. The mixture is stirred at 30 °C for 30 min; then 3-aminophenol (0.2 g) is added, and stirring is continued for 30 min; then, 0.36 mL of tetraethyl orthosilicate (TEOS) and formaldehyde (37 wt%, 0.28 mL) are added to the mixture, and the mixture is stirred at 30 °C for 5 h; the resin / silica nanocomposite particles are washed with ethanol and water 3 times or more, and centrifuged at 10000 rpm for 10 min; finally, the mixture is calcined in air from room temperature to 550 °C at a heating rate of 1 °C / min, and the calcination time is 6 h to obtain hollow mesoporous silica particles (HMPS).

[0033] Furthermore, the specific preparation process of amino- and silane-modified mesoporous silica spheres is as follows: The HMSP sample (0.5 g) is immersed in a toluene solution (10 mL), and then an alkyl group (octyltrichlorosilane (OTCS), 119.3 μL, 0.5 mmol) and various fatty amine modifiers (APTMS, 13.5 μL, 75 μmol; NPED, 17.6 μL, 75 μmol; NQ-62, 18.0 μL, 75 μmol) are introduced to obtain modified HMSP samples, which are HMSP-N 1 / C8, HMSP-N2 / C8, and HMSP-N3 / C8; The mixture was sonicated for 10 minutes to ensure homogeneity and then continuously shaken in a constant-temperature shaker at 220 rpm for 4 hours. The modified HMSP samples were separated by a purification process involving multiple (three or more) cycles of ethanol washing and centrifugation at 10,000 rpm for 10 minutes. The contact angle after modification is as Figure 2 shown.

[0034] The above-mentioned amino- and silane-coupling-agent-modified hollow mesoporous SiO2 nanospheres are about 500 nm in size, have a mesoporous and hollow structure, and from Figure 3 it can be seen that the pore size of the hollow mesoporous SiO2 nm spheres remains unchanged before and after modification, with a size of 10 - 20 nm.

[0035] In the following examples, the specific process for preparing immobilized phospholipase by the adsorption method is as follows: The modified particles (0.2 g) were dispersed in the enzyme solution (10 mL, 4.6 mg / mL), then sonicated for 5 min, and then placed in a shaker at 220 rpm at a temperature of 30 °C for 2 h. The obtained immobilized enzyme products, named PLD@HMSP-N1 / C8, PLD@HMSP-N2 / C8, and PLD@HMSP-N3 / C8, were then freeze-dried for subsequent use.

[0036] To better understand the present invention, the following examples are used to further specifically illustrate the present invention, but it should not be construed as a limitation of the present invention. For those skilled in the art, some non-essential improvements and adjustments made based on the above-mentioned invention content are also considered to fall within the protection scope of the present invention.

[0037] Example 1

[0038] 0.4 mL of ethyl butyrate solution of PC (10 mg / mL), 0.6 mL of PBS solution of L-serine (1 M, pH 6), and 10 mg of immobilized enzyme PLD@HMSP-N3 / C8 were mixed evenly, and a Pickering emulsion was formed by homogenization at 20,000 rpm for 1 minute. The water-in-oil type had an average particle size range of 87 μm and a specific surface area range of 669.6 cm 2 , and the reaction was carried out at 40 °C for 20 min. After sampling, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce the coalescence of emulsion droplets to achieve rapid separation of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a mixed solvent of chloroform / methanol (volume ratio 2:1), and the organic solvents were removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0039] After detection, the conversion rate reached 89%. Its fatty acid composition is as follows: C16:0 (34.5% ± 1.35%), C18:0 (9.5% ± 0.45%), C18:1 (28.6% ± 1.08%), C18:2 (15.3% ± 0.6%), C20:0 (4.7% ± 0.17%), C22:6 (1.4% ± 0.17%), and others (6.0% ± 3.49%).

[0040] Example 2

[0041] Dissolve PC in ethyl butyrate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). Homogenize at 20000 rpm for 1 minute to form Pickering emulsion, water-in-oil type, with an average particle size range of 88.9 μm and a specific surface area range of 638.4 cm 2 , and the reaction was carried out in a water bath at 50 °C for 20 min. Each experimental condition was carried out 3 times, and 3 repeated experiments were carried out for each group of conditions. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce droplet coalescence to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol / n-hexane (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0042] After detection, the conversion rate reached 89%.

[0043] Example 3

[0044] Dissolve PC in ethyl butyrate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). Homogenize at 20000 rpm for 1 minute to form Pickering emulsion, water-in-oil type, with an average particle size range of 82.7 μm and a specific surface area range of 612.5 cm 2 , and the reaction was carried out in a water bath at 60 °C for 30 min. Each experimental condition was carried out 3 times, and 3 repeated experiments were carried out for each group of conditions. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce droplet coalescence to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol / n-hexane (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0045] After detection, the conversion rate reached 45%.

[0046] Example 4

[0047] PC was dissolved in ethyl acetate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). A Pickering emulsion was formed by homogenization at 20000 rpm for 1 minute. It was of the water-in-oil type, with an average particle size range of 83.1 μm and a specific surface area range of 619.3 cm 2 , The reaction was carried out in a water bath at 40 °C for 20 min. Each experimental condition was performed 3 times, and 3 replicate experiments were carried out for each set of conditions. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce droplet coalescence to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0048] The conversion rate was detected to reach 66%.

[0049] Example 5

[0050] PC was dissolved in ethyl propionate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). A Pickering emulsion was formed by homogenization at 20000 rpm for 1 minute. It was of the water-in-oil type, with an average particle size range of 85.2 μm and a specific surface area range of 649.2 cm 2 , The reaction was carried out in a water bath at 40 °C for 20 min. Each experimental condition was performed 3 times, and 3 replicate experiments were carried out for each set of conditions. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce droplet coalescence to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0051] The conversion rate was detected to reach 78%.

[0052] Example 6

[0053] PC was dissolved in propyl propionate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). A Pickering emulsion was formed by homogenization at 20000 rpm for 1 minute. It was of the water-in-oil type, with an average particle size range of 88.5 μm and a specific surface area range of 657.3 cm 2, The reaction was carried out in a 40 °C water bath for 20 min. Each experimental condition was performed 3 times, and each set of conditions was replicated 3 times. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce the coalescence of emulsion droplets to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0054] After detection, the conversion rate reached 41%.

[0055] Example 7

[0056] PC was dissolved in ethyl valerate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). Homogenization was carried out at 20000 rpm for 1 minute using a homogenizer to form a Pickering emulsion, water-in-oil type, with an average particle size range of 83.1 μm and a specific surface area range of 597.2 cm 2 , The reaction was carried out in a 40 °C water bath for 40 min. Each experimental condition was performed 3 times, and each set of conditions was replicated 3 times. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce the coalescence of emulsion droplets to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0057] After detection, the conversion rate reached 68%.

[0058] Example 8

[0059] PC was dissolved in ethyl hexanoate (10 mg / mL, 0.4 mL). The reaction mixture was initiated by L-serine (1 M, 0.6 mL) and immobilized enzyme (1 wt%, 10 mg). Homogenization was carried out at 20000 rpm for 1 minute using a homogenizer to form a Pickering emulsion, water-in-oil type, with an average particle size range of 85.6 μm and a specific surface area range of 626.6 cm 2 , The reaction was carried out in a 40 °C water bath for 60 min. Each experimental condition was performed 3 times, and each set of conditions was replicated 3 times. After the reaction, low-temperature centrifugation (4 °C, 5000 rpm, 10 min) was used to induce the coalescence of emulsion droplets to achieve rapid stratification of the oil and water phases. The demulsified aqueous phase (containing PS product) was extracted 3 times with a chloroform / methanol (volume ratio 2:1) mixed solvent, and the organic solvent was removed by vacuum distillation (40 - 50 °C, 0.1 MPa) to obtain PS.

[0060] After detection, the conversion rate reaches 53%.

[0061] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing phosphatidylserine by interfacial enzyme catalysis, characterized in that, It includes the following steps: (1) Preparation of immobilized phospholipase D: Hollow mesoporous silica particles (HMSP) were prepared by sol-gel method combined with template sacrificial method, and the surface of HMSP was modified with dual functional groups of nitrogen-containing alkylamine and long-chain alkyl to regulate the hydrophilicity and hydrophobicity of the carrier surface; Phospholipase D (PLD) was immobilized in the carrier pores by adsorption method to obtain immobilized enzymes PLD@HMSP-N1 / C8, PLD@HMSP-N2 / C8, PLD@HMSP-N3 / C8; (2) Preparation of PS in interfacial enzyme catalysis system: The immobilized enzyme was used as both catalyst and emulsifier to stabilize the W / O Pickering emulsion composed of ethyl butyrate, ethyl acetate, ethyl propionate, propyl propionate, ethyl valerate or ethyl caproate and phosphate buffer solution, citrate buffer solution, citric acid / sodium citrate buffer solution; Phosphatidylcholine (PC) and serine (L-Ser) were dissolved in organic solvent and phosphate buffer solution respectively, and phosphatidylserine (PS) was prepared by reaction; (3) Separation and purification of PS: After the reaction, low-temperature centrifugation was used to induce the aggregation of emulsion droplets to achieve rapid separation of oil and water phases; The separated aqueous phase (containing PS product) was extracted with a mixed solvent, and then the organic solvent was removed by vacuum distillation to obtain PS.

2. The method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that, In step (1), the carrier HMSP was synthesized by a one-pot sol-gel method using tetraethyl orthosilicate as the silicon source and 3-aminophenol / formaldehyde resin as the template. The template was removed by high-temperature calcination to obtain hollow mesoporous silica particles, named HMPS.

3. The method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that In step (1), the dual functional group modification combinations include (3-aminopropyl) trimethoxysilane and (APTMS), N-[3-(trimethoxysilyl)propyl]ethylenediamine (NPED) or 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (NQ-62) and n-octyltrichlorosilane (OTCS).

4. A method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that, In step (1), the main sources of the phospholipase D include Streptomyces, Bacillus, Pseudomonas, Actinomyces, Clostridium, Lactobacillus, Enterobacter, Salmonella, Escherichia coli, Staphylococcus.

5. A method for preparing phosphatidylserine by interfacial enzyme catalysis according to any one of claims 1-4, characterized in that, In step (1), the protein concentration of the phospholipase D enzyme solution is 4.6 mg / mL; The solid-liquid ratio of the carrier to the enzyme solution is 10:1 - 50:1 (mg / mL); The immobilization time is 30 - 150 min, and the temperature is 20 - 40 °C; The pH of the phosphate buffer solution is 4.0 - 8.

0.

6. The method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that, In step (2), the volume ratio of the oil phase to the water phase in the interfacial enzyme catalysis system is 3:7 - 7:3, the addition amount of the immobilized enzyme is 0.5 wt% - 2.0 wt%, the PC concentration is 10 - 50 mg / mL, the L-Ser concentration is 1 - 4.0 mol / L, the pH of the reaction system is 4 - 8, the reaction temperature is 30 - 70 °C, and the reaction time is 20 - 60 min.

7. A method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that, In step (3), the mixed solvent is a chloroform / methanol solvent with a volume ratio of 2:

1.

8. The method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, wherein In step (3), the temperature of the low-temperature centrifugation is 4 °C, the rotation speed is 5000 rpm, and the time is 10 min.

9. A method for preparing phosphatidylserine by interfacial enzyme catalysis according to claim 1, characterized in that, In step (3), the temperature of the vacuum distillation is 40 - 50 °C and the pressure is 0.1 MPa.

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