Application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA

Abstract

The present invention provides an application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA. The specific process is as follows: cobalt nitrate hexahydrate and 2-methylimidazole are respectively dissolved in anhydrous methanol to obtain a cobalt nitrate hexahydrate solution and a 2-methylimidazole solution. The 2-methylimidazole solution is added dropwise to the cobalt nitrate hexahydrate solution, stirred and reacted, allowed to stand, and dried to obtain ZIF-67 nanomaterials. The ZIF-67 nanomaterials are suspended in a sodium phosphate buffer solution, a phospholipase A1 solution is added, the pH is adjusted, and the mixture is incubated on a shaker, centrifuged and dried to obtain ZIF-67 nanomaterial-immobilized phospholipase A1. A substrate composed of free fatty acids and soybean lecithin and the immobilized phospholipase A1 are subjected to a catalytic reaction in an organic reaction system, and phosphatidyl DHA is obtained by separation and purification. The immobilized phospholipase A1 has good stability, catalyzes the preparation of phosphatidyl DHA, and the DHA incorporation rate is as high as 51.04%.

Description

Technical Field

[0001] This application belongs to the technical field of enzyme engineering and biocatalysis. Specifically, it relates to the application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA. Background Art

[0002] Phospholipids are the main components of cell membranes and have functions such as anti-atherosclerosis and lipid-lowering. Docosahexaenoic acid (DHA) contributes to visual development, enhances immunity, improves heart health, and promotes intellectual development. However, its high degree of unsaturation makes it prone to oxidation. Binding DHA to phospholipids not only improves its oxidative stability but also enhances its absorption in the human body, and at the same time has the dual effects of phospholipids and DHA. In recent years, there have been many studies on the preparation of phosphatidyl DHA using enzymatic methods. For example, patent CN110438171A discloses an in vitro enzymatic catalytic conversion technology to achieve the in vitro conversion and preparation of lecithin-type DHA. Patent CN104531790A discloses a reaction system in which anionic / cationic surfactants are dissolved in a hydrophobic alkane solvent, and phosphatidyl DHA is obtained by catalyzing the reaction with phospholipase in this system.

[0003] As an important industrial enzyme for producing phosphatidyl DHA, phospholipase has the characteristics of high selectivity, mild catalytic conditions, and environmental friendliness. Although phospholipase has great potential in industrial applications, most free phospholipases have problems such as high preparation cost, poor operational stability, easy inactivation in organic solvents, and easy caking. These factors greatly limit its application in large-scale production. In contrast, immobilized phospholipase can significantly improve its stability and catalytic efficiency, and at the same time can also improve the enzyme recovery rate and facilitate repeated use. As a nanoscale carrier for immobilizing phospholipase A1, ZIF-67 (zeolitic imidazolate framework) has attracted extensive attention in the field of biocatalysis in recent years. ZIF-67 has a high specific surface area, which provides abundant binding sites for the loading of phospholipase A1, thus improving the catalytic efficiency of the enzyme. Summary of the Invention

[0004] The present invention provides an application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA to solve the above-mentioned defects and deficiencies existing in the prior art.

[0005] To solve the above technical problems, the present invention provides an application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA, which is characterized in that:

[0006] The process of in-situ synthesis of immobilized phospholipase A1 is as follows: cobalt nitrate hexahydrate and 2-methylimidazole are respectively dissolved in anhydrous methanol to obtain a cobalt nitrate hexahydrate solution and a 2-methylimidazole solution. The 2-methylimidazole solution is added dropwise to the cobalt nitrate hexahydrate solution, stirred, reacted, allowed to stand, and dried to obtain ZIF-67 nanomaterials. The ZIF-67 nanomaterials are suspended in a sodium phosphate buffer solution, a phospholipase A1 solution is added, the pH is adjusted, and the mixture is incubated on a shaker, centrifuged, and dried to obtain ZIF-67 nanomaterial-immobilized phospholipase A1;

[0007] The steps of the application are as follows: a substrate composed of free fatty acid and soybean lecithin and immobilized phospholipase A1 are added to an organic reaction system for catalytic reaction, and after the reaction is completed, separation and purification are carried out to obtain phosphatidyl DHA.

[0008] Furthermore, the concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 13 g / L - 16 g / L, the concentration of 2-methylimidazole dissolved in anhydrous methanol is 18 g / L - 22 g / L, and the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 0.8 - 1.2:1.

[0009] Furthermore, the mass ratio of the ZIF-67 nanomaterials to phospholipase A1 is 200:58 - 290.

[0010] Furthermore, the mass ratio of free fatty acid, soybean lecithin, and immobilized phospholipase A1 is 2.5:0.25:0.15 - 0.25.

[0011] Furthermore, the immobilization time in the process of in-situ synthesis of immobilized phospholipase A1 is 4 - 20 h.

[0012] Furthermore, the immobilization temperature in the process of in-situ synthesis of immobilized phospholipase A1 is 10°C - 50°C.

[0013] Furthermore, the pH in the process of in-situ synthesis of immobilized phospholipase A1 is 2 - 10.

[0014] The beneficial technical effects achieved by the present invention: The present invention provides an application of immobilized phospholipase A1 in the preparation of phosphatidyl DHA. The immobilized phospholipase A1 prepared using ZIF-67 nanocarriers has good stability, catalyzes the preparation of phosphatidyl DHA, and the DHA incorporation rate is as high as 51.04%. Description of the Drawings

[0015] Figure 1 This is the ZIF-67 nanomaterial powder prepared in the example of the present invention;

[0016] Figure 2 This is the SEM image of the ZIF-67 nanomaterials prepared in the example of the present invention;

[0017] Figure 3SEM and EDS diagrams of the immobilized phospholipase ZIF-67@PLA1 prepared in the embodiments of the present invention;

[0018] Figure 4 Diagram of the phospholipid DHA product prepared in the embodiments of the present invention;

[0019] Figure 5 Effect of different enzyme addition amounts on the immobilization rate, relative enzyme activity and phospholipid DHA incorporation rate during the immobilization reaction of the present invention;

[0020] Figures 6 - 10 Gas chromatogram of the effect of different enzyme addition amounts on the immobilization reaction in the embodiments of the present invention; among them, Figure 6 indicates that the volume of phospholipase A1 is 2 mL, Figure 7 is the volume of phospholipase A1 being 4 mL, Figure 8 is the volume of phospholipase A1 being 6 mL, Figure 9 is the volume of phospholipase A1 being 8 mL, Figure 10 is the volume of phospholipase A1 being 10 mL;

[0021] Figure 11 Effect of different pH values on the immobilization rate, relative enzyme activity and phospholipid DHA incorporation rate during the immobilization reaction of the present invention;

[0022] Figures 12 - 16 Gas chromatogram of the effect of different pH values on the immobilization reaction in the embodiments of the present invention; among them, Figure 12 the pH value of [[ ]] is taken as 2, Figure 13 the pH value of [[ ]] is taken as 4, Figure 14 the pH value of [[ ]] is taken as 6, Figure 15 the pH value of [[ ]] is taken as 8, Figure 16 the pH value of [[ ]] is taken as 10;

[0023] Figure 17 Effect of different immobilization times on the immobilization rate, relative enzyme activity and phospholipid DHA incorporation rate during the immobilization reaction of the present invention;

[0024] Figures 18 - 22 Gas chromatogram of the effect of different immobilization times on the immobilization reaction in the embodiments of the present invention; among them, Figure 18 the immobilization time of [[ ]] is 4 h, Figure 19 the immobilization time of [[ ]] is 8 h, Figure 20 the immobilization time of [[ ]] is 12 h, Figure 21 the immobilization time of [[ ]] is 16 h, Figure 22 the immobilization time of [[ ]] is 20 h;

[0025] Figure 23 Effect of different immobilization temperatures on the immobilization rate, relative enzyme activity and phospholipid DHA incorporation rate during the immobilization reaction of the present invention;

[0026] Figures 24 - 28 Gas chromatogram showing the effect of different immobilization temperatures on the immobilization reaction in the embodiments of the present invention; wherein, Figure 24 The immobilization temperature of [[ ]] is 10°C, Figure 25 The immobilization temperature of [[ ]] is 20°C, Figure 26 The immobilization temperature of [[ ]] is 30°C, Figure 27 The immobilization temperature of [[ ]] is 40°C, Figure 28 The immobilization temperature of [[ ]] is 50°C;

[0027] Figure 29 Shows the effect of different dosages of immobilized phospholipase A1 on the incorporation rate of phospholipid DHA in the present invention;

[0028] Figures 30 - 33 Gas chromatogram showing the effect of different dosages of immobilized phospholipase A1 on the incorporation rate of phospholipid DHA in the present invention, wherein, Figure 30 The dosage of immobilized phospholipase A1 of [[ ]] is 50 mg, Figure 31 The dosage of immobilized phospholipase A1 of [[ ]] is 150 mg, Figure 32 The dosage of immobilized phospholipase A1 of [[ ]] is 250 mg, Figure 33 Is free enzyme, and the dosage is 150 mg. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and cannot be used to limit the protection scope of the present invention.

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] The reagents and manufacturers used in the following embodiments are shown in Table 1:

[0032] Table 1

[0033] Reagent Name Manufacturer n - Hexane Shanghai Macklin Biochemical Co., Ltd. Soybean Lecithin Anhui Zhongchuang Phospholipid Technology Co., Ltd. Free Fatty Acid Hunan Wanquan Yuxiang Biotechnology Co., Ltd. Phospholipase A1 Genencor Bioengineering Co., Ltd.

[0034] Example 1 Preparation of immobilized phospholipase A1

[0035] I. Preparation of ZIF-67 nanomaterials: Cobalt nitrate hexahydrate and 2-methylimidazole are respectively dissolved in anhydrous methanol to obtain a cobalt nitrate hexahydrate solution and a 2-methylimidazole solution. After stirring at room temperature for 20 min, the 2-methylimidazole solution is added dropwise to the cobalt nitrate hexahydrate solution, and stirring is continued for 1 h. The mixture is allowed to stand at room temperature for 24 h, and the resulting mixture is centrifuged at 8000 r / min. The obtained precipitate is washed once with methanol and then twice with deionized water, and dried in an oven at 50 °C to obtain ZIF-67 nanomaterials. The concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 13 g / L - 16 g / L, the concentration of 2-methylimidazole dissolved in anhydrous methanol is 18 g / L - 22 g / L, and the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 0.8 - 1.2:1. Among them:

[0036] ZIF-67 nanomaterial 1: The concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 13 g / L, the concentration of 2-methylimidazole dissolved in anhydrous methanol is 18 g / L, and the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 0.8:1;

[0037] ZIF-67 nanomaterial 2: The concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 15 g / L, the concentration of 2-methylimidazole dissolved in anhydrous methanol is 20 g / L, and the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 1:1;

[0038] ZIF-67 nanomaterial 3: The concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 16 g / L, the concentration of 2-methylimidazole dissolved in anhydrous methanol is 22 g / L, and the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 1.2:1.

[0039] Samples of ZIF-67 nanomaterial 1 to ZIF-67 nanomaterial 3 are respectively as shown in Figure 1 1A - 1C, and the SEM images are as shown in Figure 2 2A - 2C, and the BET analysis results are shown in Table 2.

[0040] Table 2

[0041] Sample <![CDATA[Surface area (m 2 / g)]]> <![CDATA[Total pore volume (cm 3 / g)]]> Average Pore Diameter (nm) ZIF - 67 Nanomaterial 1 988.1201 m² / g 0.493143 cm³ / g 11.3621 nm ZIF - 67 Nanomaterial 2 1044.3003 m² / g 0.542747 cm³ / g 10.7641 nm ZIF - 67 Nanomaterial 3 1136.2997 m² / g 0.570637 cm³ / g 9.6213 nm

[0042] From Figure 2 and Table 2, it can be seen that: The above-prepared ZIF-67 nanomaterials all have a very high surface area and pore volume, and the pore radius is about 10 nm. Among them, the surface area of ZIF-67 nanomaterial 2 is 1044.3003 m 2 / g, and the pore diameter is 10.7641 nm. Therefore, ZIF-67 nanomaterial 2 is selected for the immobilization reaction of phospholipase A1 in the subsequent examples.

[0043] II. Preparation of immobilized phospholipase A1: ZIF-67 nanomaterial II was added to phosphate buffer for suspension, and then phospholipase A1 solution was added, and the pH value was adjusted to 6. Then, the mixture was incubated in a shaker at 20 °C for 12 h. After incubation, the immobilized enzyme was separated by centrifugation and washed 1-2 times with buffer to remove residual free enzyme. The obtained wet immobilized enzyme was then dried in an oven at 30 °C to obtain immobilized phospholipase ZIF-67@PLA, which was stored at 8 °C for subsequent enzyme activity assay; among them, the addition amount of ZIF-67 nanomaterial II was 200 mg, the phosphate buffer was disodium hydrogen phosphate solution, the concentration of phospholipase A1 solution was 29 mg / mL, the added volume was 6 mL, the volume ratio of phosphate buffer to phospholipase A1 was 1:1, and the SEM and EDS diagrams of immobilized phospholipase ZIF-67@PLA are as Figure 3 shown.

[0044] It can be seen from Figure 3 that: Figure 2 The SEM diagram shown is that the pure ZIF material has a cubic surface morphology, which is a typical feature of ZIF-67. Figure 3 The SEM diagram of shows that the nanomaterial becomes round after loading phospholipase A1, and the cubic structure is no longer obvious. This may be due to the change in the particle surface caused by the loading of the enzyme, and this morphological change may help to enhance the catalytic activity and stability of the material. Figure 3 The EDS image of shows the element distribution and distribution fraction on the surface of ZIF-67 nanomaterial after immobilizing phospholipase A1. Among them, the C element is 37.83%, the N element is 5.10%, the O element is 31.01%, and the Co element is 26.06%. These element distributions reflect the modification of phospholipase A1 on the surface of ZIF-67 nanomaterial II. In summary, the SEM and EDS data show the morphological and compositional changes of ZIF-67 material during enzyme loading and surface modification.

[0045] III. Preparation of phospholipid DHA: The substrate composed of free fatty acid and soy lecithin and immobilized phospholipase ZIF-67@PLA were sequentially added to a reverse micelle system (containing a certain proportion of buffer) prepared with n-hexane and sodium dioctyl sulfosuccinate (where the concentration of sodium dioctyl sulfosuccinate relative to n-hexane is 22.2 g / L), and the catalytic reaction was carried out in a shaker. After the reaction, the immobilized phospholipase ZIF-67@PLA was removed by filtration, the filtrate was taken out, and then acetone was added for elution. The insoluble matter was phospholipid DHA, and it was centrifuged at 7000 rpm for 5 min and the operation was repeated 5 times. Vacuum concentration and drying were carried out to obtain dry phospholipid DHA powder, as Figure 4 shown.

[0046] Among them, the mass of free fatty acids is 2.5g, the mass of soybean lecithin is 0.25g, the addition amount of immobilized phospholipase ZIF-67@PLA is 150mg, the addition amount of buffer in the reverse micelle system is 4.472%, the catalytic reaction is carried out on a shaker, the reaction temperature is 40℃, the reaction time is 24h, the rotation speed is 200rpm; the vacuum concentration temperature is 45℃.

[0047] Experimental Example 1 Effect of Phospholipase A1 Immobilized on Different Carriers on the Preparation of Phospholipid DHA

[0048] The ZIF-67 nanomaterial II in the preparation process of immobilized phospholipase A1 was replaced with other types of carriers, and the data such as the debt ratio to phospholipase A1 were investigated, as shown in Table 3.

[0049] Table 3

[0050] Carrier Temperature (°C) Immobilization Rate (%) Loading Rate (mg / g) Enzyme Activity (U / g) ZIF - 67 Nanomaterial 2 20 69 619.8 2785.714 Duolite A568 Resin 50 / / / DA - 201 Resin 50 / 85 / Lewatit VPOC1600 55 79.5 147.9 <![CDATA[6.7×10 −3 > Microfluidic Support 55 81.2 45.7 415.9 PVA - alginate beads 58 62.93% / 88.78 D380 Resin 55 15 36.96 348.42

[0051] From Table 3, it can be seen that the loading rate of phospholipase A1 loaded by ZIF-67 nanomaterial II as a carrier is as high as 619.8 mg / g, and the enzyme activity is still maintained at 2785.714 U / g. Table 3 shows that Lewatit VPOC1600, microfluidic support, and PVA-alginate beads all have high fixation rates, but the enzyme activity and loading rate are sharply reduced. ZIF-67 nanomaterial II was selected to prepare phospholipid DHA, and the gas chromatogram was used to calculate the incorporation rate of phospholipid DHA / EPA, and the results are shown in Table 4.

[0052] Table 4

[0053] Carrier Immobilized Enzyme Enzyme Addition Amount (g) Incorporation Rate of Phospholipid DHA / EPA (%) ZIF - 67 Nanomaterial 2 0.1 51.04 Duolite A568 Resin 0.2 35 DA - 201 Resin 1.5 59 Lewatit VPOC1600 0.9 16.7 Microfluidic Support / 54.3 PVA - alginate beads / / D380 Resin 2 30.7

[0054] Experimental Example 2 Effect of the Preparation Conditions of Immobilized Phospholipase A1 on the Obtaining Phospholipid DHA

[0055] 1. Amount of phospholipase A1: Immobilized phospholipase A1 was prepared according to the method of Example 1, wherein the volume of phospholipase A1 was 2, 4, 6, 8, and 10 mL, respectively. The immobilized phospholipase A1 was obtained, and the effect on the immobilization rate and enzyme activity of the phospholipase was as follows: Figure 5 As shown in 5A in the figure: As the amount of phospholipase A1 added increases, the immobilization rate and relative enzyme activity of the enzyme show an upward trend. When the amount added is 6 mL, it reaches the maximum. When the amount is continued to be added, it begins to decrease. Therefore, the amount of phospholipase A1 added is determined to be 6 mL. Phospholipid DHA was prepared according to the preparation method of phospholipid DHA in Example 1, and the incorporation rate of phospholipid DHA was calculated using the gas chromatogram as shown in Figure 5 As shown in Figure 5B: The incorporation rate of phospholipid DHA is the highest when the amount of phospholipase A1 is 6 mL, reaching 40.92%. The gas chromatograms are as follows: Figures 6 - 10 shown.

[0056] 2. pH value: Immobilized phospholipase A1 was prepared according to the method of Example 1 at pH values of 2, 4, 6, 8, and 10 respectively to obtain immobilized phospholipase A1. The effects on the immobilization rate and enzyme activity of phospholipase are shown in Figure 11 Figure 11A as follows: When the pH is 6, the immobilization rate and relative enzyme activity are the highest. Phospholipid DHA was prepared according to the method for preparing phospholipid DHA in Example 1. The incorporation rate of phospholipid DHA is shown in Figure 11 Figure 11B as follows: When the pH is 6, the incorporation rate of DHA is the highest. The gas chromatograms are shown in Figures 12 - 16 Figure 11C respectively.

[0057] 3. Immobilization time: Immobilized phospholipase A1 was prepared according to the method of Example 1 with immobilization times of 4 h, 8 h, 12 h, 16 h, and 20 h respectively to obtain immobilized phospholipase A1. The effects on the immobilization rate and enzyme activity of phospholipase are shown in Figure 17 Figure 17A as follows. Phospholipid DHA was prepared according to the method for preparing phospholipid DHA in Example 1. The incorporation rate of DHA is shown in Figure 17 Figure 17B as follows, and the gas chromatograms are shown in Figures 18 - 22 Figure 17C respectively.

[0058] 4. Immobilization temperature: Immobilized phospholipase A1 was prepared according to the method of Example 1 at immobilization temperatures of 10 °C, 20 °C, 30 °C, 40 °C, and 50 °C respectively to obtain immobilized phospholipase A1. The effects on the immobilization rate and enzyme activity of phospholipase are shown in Figure 23 Figure 23A as follows. Phospholipid DHA was prepared according to the method for preparing phospholipid DHA in Example 1. The incorporation rate of DHA is shown in Figure 23 Figure 23B as follows, and the gas chromatogram is shown in Figures 24 - 28 Figure 23C respectively.

[0059] 5. Dosage of immobilized phospholipase A1: Phospholipid DHA was prepared according to the method for preparing phospholipid DHA in Example 1. The dosages of immobilized phospholipase A1 were 50 mg, 150 mg, and 250 mg, and the dosage of free phospholipase A1 was 150 mg. The incorporation rate of DHA is shown in Figure 29 Figure 33 as follows, and the gas chromatograms are shown in Figures 30 - 33 Figure 34 respectively. It can be seen from Figure 29 Figure 35 that when the dosage of immobilized phospholipase A1 is different, the incorporation rate of DHA is also different. Among them, when the dosage is 150 mg, the incorporation rate of DHA is the highest, which is more than 51%. Compared with the incorporation rate of 30% of free phospholipase, it is increased by 20%.

[0060] The DHA incorporation rate was calculated by gas chromatography: Weigh 0.1 g of phospholipid-DHA into a 10 mL volumetric flask, add 3 mL of 0.5 M KOH-methanol solution, react in a constant temperature water bath at 65 °C for 17 min, take it out and cool to room temperature, then add 3 mL of boron trifluoride ether-methanol (3:7, v / v), react in a constant temperature water bath at 65 °C for 7 min, take it out and cool to room temperature; add 2 mL of saturated NaCl solution and mix well; then add 2 mL of n-hexane for extraction for 30 min; take 1 mL of the supernatant and filter it through a 0.2 µm organic membrane into a gas-phase vial for testing. Gas chromatograph: Shimadzu GC-2010; chromatographic column: model DB-23 (60 m × 0.25 mm × 0.25 µm); carrier gas: nitrogen; detector: FID; split ratio: 35 / 1; inlet temperature: 250 °C; detector temperature: 280 °C; column temperature programming: initial temperature 100 °C, increase to 200 °C at 25 °C / min, then increase to 240 °C at 2 °C / min, and finally hold at 240 °C for 7 min, the total analysis time is 35 min; air flow rate 400 mL / min; hydrogen flow rate: 40 mL / min; column flow rate: 3.0 mL / min; tail blow flow rate: 30 mL / min; detector temperature: 280 °C; injection volume: 1 µL.

[0061] The present invention has been disclosed in the preferred embodiments above, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitution or equivalent transformation schemes fall within the protection scope of the present invention.

Claims

1. Use of immobilized phospholipase A1 in the preparation of phosphatidyl DHA, characterized in that: The preparation process of the immobilized phospholipase A1 is as follows: Cobalt nitrate hexahydrate and 2-methylimidazole are respectively dissolved in anhydrous methanol to obtain a cobalt nitrate hexahydrate solution and a 2-methylimidazole solution. The 2-methylimidazole solution is added dropwise to the cobalt nitrate hexahydrate solution, stirred and reacted, allowed to stand, and dried to obtain ZIF-67 nanomaterials. The ZIF-67 nanomaterials are suspended in a sodium phosphate buffer solution, a phospholipase A1 solution is added, the pH is adjusted to 6, and incubated in a shaker for 12 hours, centrifuged and dried to obtain ZIF-67 nanomaterial-immobilized phospholipase A1; the concentration of cobalt nitrate hexahydrate dissolved in anhydrous methanol is 15 g / L, and the concentration of 2-methylimidazole dissolved in anhydrous methanol is 20 g / L; the volume ratio of the 2-methylimidazole solution to the cobalt nitrate hexahydrate solution is 1:1; the mass ratio of the ZIF-67 nanomaterials to phospholipase A1 is 200:58-290; The steps for preparing phosphatidyl DHA using the above immobilized phospholipase A1 are as follows: A substrate composed of free fatty acids and soy lecithin and the immobilized phospholipase A1 are added to an organic reaction system for catalytic reaction, and after the reaction is completed, separation and purification are carried out to obtain phosphatidyl DHA; the mass ratio of free fatty acids, soy lecithin and immobilized phospholipase A1 is 2.5:0.25:0.

15.

2. Use of the immobilized phospholipase A1 according to claim 1 in the preparation of phospholipid DHA, characterized in that: The incubation temperature during the synthesis of the immobilized phospholipase A1 is 10°C - 50°C.

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