Immobilized phospholipase D and application thereof in preparation of DHA-PE

Abstract

The invention discloses immobilized phospholipase D. The immobilized phospholipase D is prepared by the following method: phospholipase D-Sp-256-A409C is loaded on D301 alkalescent styrene anion resin, and the enzyme loading ratio is 10-120 mg / g. According to the application of the immobilized phospholipase D in preparation of DHA-PE, DHA-phosphatidylcholine and ethanolamine hydrochloride are taken as substrates, and transesterification reaction is carried out under the catalytic action of the immobilized phospholipase D to synthesize DHA-PE. According to the method, the D301 alkalescent styrene anion resin is used as a carrier to adsorb phospholipase D-Sp-256-A409C, the protein adsorption capacity is high, the transesterification activity is high, the enzyme activity recovery rate is high, and the method can be used for efficiently preparing DHA-PE. The research of the invention has important significance for industrial preparation of DHA-PE.

Description

Technical Field

[0001] This invention relates to an immobilized phospholipase D and its application in the preparation of DHA-PE, belonging to the field of immobilized enzyme technology. Background Technology

[0002] Phospholipase D (PLD), or phosphatidylcholine hydrolase, is a highly efficient phospholipid modification catalyst capable of selectively synthesizing various rare phospholipids. In previous studies, this invention screened a phospholipase D-Sp-256-A409C through mutagenesis. This enzyme exhibits a substrate conversion rate of up to 91.2% and a transesterification activity of 115.0 μg / h / mg in the preparation of DHA-phosphatidylethanolamine (DHA-PE), demonstrating broad application prospects. The relevant details are described in Chinese Invention Patent CN118895264 A.

[0003] However, when using phospholipase D to catalyze the synthesis of rare phospholipids, free enzyme proteins exhibit common protein properties such as sensitivity, instability, and easy inactivation, and also suffer from low operational stability and difficulty in recycling, significantly limiting their industrial applications. In this context, enzyme immobilization is an effective approach to expand its applicability, improve operational stability, enhance catalytic performance, and enable enzyme recycling. Currently, there are no reports on the immobilization of phospholipase D-Sp-256-A409C. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an immobilized phospholipase D and its application in the preparation of DHA-PE.

[0005] This invention is achieved through the following technical solution: An immobilized phospholipase D is prepared by loading phospholipase D-Sp-256-A409C onto a D301 weakly basic styrene-based anion exchange resin, with an enzyme loading ratio of 10–120 mg / g, preferably 60 mg / g. The amino acid sequence of the phospholipase D-Sp-256-A409C is shown below, as in SEQ ID NO.1: .

[0006] Furthermore, it was prepared by the following method: adding an enzyme solution containing phospholipase D-Sp-256-A409C and a citrate-sodium citrate buffer solution with pH 3.0-10.0 to a D301 weakly basic styrene-based anion exchange resin to make the enzyme loading ratio 10-120 mg / g; shaking adsorption for 1-6 h; filtering off the supernatant, rinsing with buffer solution, and freeze-drying to obtain the immobilized enzyme.

[0007] Furthermore, it was prepared by the following method: an enzyme solution containing phospholipase D-Sp-256-A409C was added to a D301 weakly basic styrene-based anion exchange resin, along with an equal volume of 20 mmol / L pH 5.0 citrate-sodium citrate buffer to achieve an enzyme loading ratio of 60 mg / g; the enzyme was adsorbed by shaking at 4°C and 220 r / min for 3 h; the supernatant was filtered off, the enzyme was washed with buffer, and then freeze-dried to obtain the immobilized enzyme.

[0008] Further, the D301 weakly basic styrene-based anion exchange resin was pretreated as follows: 50 g of D301 weakly basic styrene-based anion exchange resin was taken, and 150 mL of saturated sodium chloride solution was added. The resin was soaked for 18–20 hours. The sodium chloride solution was drained, and the resin was rinsed with deionized water. The resin was then soaked in 150 mL of 5% hydrochloric acid for 2–4 hours. The acid solution was drained, and the resin was rinsed with deionized water until the pH value was neutral. The resin was then soaked in 150 mL of 4% sodium hydroxide solution for 4–8 hours. The alkali solution was drained, and the resin was rinsed with deionized water until the pH value was neutral.

[0009] The application of the immobilized phospholipase D in the preparation of DHA-PE.

[0010] Furthermore, in specific applications, DHA-phosphatidylcholine (DHA-PC) and ethanolamine hydrochloride are used as substrates to synthesize DHA-PE through a transesterification reaction catalyzed by immobilized phospholipase D.

[0011] Furthermore, the method for preparing DHA-PE is as follows: the organic phase and the aqueous phase are mixed and reacted at 30-70°C, preferably at 40°C, and the reaction time is preferably 8 h; the aqueous phase is a 10 mg / mL DHA-PC solution, and the solvent is cyclopentyl methyl ether; the organic phase is: 50 mM anhydrous calcium chloride is dissolved in a 20 mmol / L citrate-sodium citrate buffer solution with pH 3.0-10.0, and 150 mg of immobilized enzyme is added; the molar ratio of ethanolamine hydrochloride to DHA-PC (i.e., the substrate molar ratio) is (5-200):1, preferably 10:1; the volume ratio of the organic phase to the aqueous phase is (1-3):(1-4), preferably 1:2.

[0012] The immobilized phospholipase D of this invention uses D301 weakly basic styrene-based anion exchange resin as a carrier to adsorb phospholipase D-Sp-256-A409C. It exhibits high protein adsorption capacity, high transesterification activity, and high enzyme activity recovery rate, making it suitable for the efficient preparation of DHA-PE. This invention optimizes the transesterification reaction conditions for DHA-PE preparation and studies its operational stability. Results show that after three reuses, the remaining enzyme activity of the immobilized phospholipase D still reaches over 90%, and after seven reuses, the remaining enzyme activity is still 84.6%. Compared to the free enzyme, the immobilized enzyme demonstrates significantly improved operational stability and catalytic performance. This research is of great significance for the industrial preparation of DHA-PE.

[0013] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0014] Figure 1Schematic diagram showing the results of protein adsorption, enzyme activity recovery rate, and relative transesterification activity of 10 resin-immobilized enzymes.

[0015] Figure 2 Schematic diagram showing the results of protein adsorption, enzyme activity recovery, and relative transesterification activity of immobilized enzymes at different adsorption times.

[0016] Figure 3 Schematic diagram showing the results of measuring the protein adsorption amount, hydrolytic enzyme activity recovery rate, and relative transesterification activity of immobilized enzymes at different pH values.

[0017] Figure 4 Schematic diagram showing the results of measuring the protein adsorption amount, hydrolytic enzyme activity recovery rate, and relative transesterification activity of immobilized enzymes at different enzyme loading ratios.

[0018] Figure 5 Scanning electron microscope (SEM) images of D301 macroporous resin and immobilized enzymes (magnification 5×10⁻⁶). 4 ), where A: D301 macroporous resin; B: immobilized enzyme.

[0019] Figure 6 Fourier transform infrared spectra of D301 macroporous resin, immobilized enzyme, and free enzyme, where a: D301 macroporous resin; b: immobilized enzyme; c: free enzyme.

[0020] Figure 7 Schematic diagram showing the results of measuring the relative transesterification activities of immobilized and free enzymes at different substrate molar ratios.

[0021] Figure 8 Schematic diagram showing the results of measuring the relative transesterification activities of immobilized and free enzymes at different pH values.

[0022] Figure 9 Schematic diagram showing the results of measuring the relative transesterification activities of immobilized and free enzymes at different reaction temperatures.

[0023] Figure 10 Schematic diagram showing the results of measuring the relative transesterification activities of immobilized and free enzymes at different organic phase / water ratios.

[0024] Figure 11 Schematic diagram of the relative enzyme activity assay results after 1 to 7 reuses of immobilized enzyme. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0026] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0027] Experiment 1: Study on the preparation conditions of immobilized phospholipase D In previous studies, this invention screened and mutated a phospholipase D-Sp-256-A409C, which can efficiently prepare DHAPE. To facilitate its industrial application, this experiment immobilized it. Macroporous resin, as an immobilization carrier, has advantages such as low cost, simple immobilization method, mild conditions, and high enzyme activity retention. Therefore, this experiment used macroporous resin as the immobilization material, and optimized the immobilization time, pH, and enzyme loading ratio. This experiment also systematically studied the application of resin-immobilized phospholipase D in the synthesis of DHAPE, and investigated its operational stability.

[0028] 1.1 Experimental Methods 1.1.1 Resin Pretreatment Ten types of resins (AB-8 macroporous adsorption resin, 724 macroporous weakly acidic acrylic cation exchange resin, D101 macroporous adsorption resin, D113 macroporous weakly acidic cation exchange resin, H103 macroporous adsorption resin, D301 weakly basic styrene-based anion exchange resin, DA201 macroporous adsorption resin, HPD-826 macroporous adsorption resin, NKA-9 macroporous adsorption resin, and XAD-1180N macroporous adsorption resin, all purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were pretreated as follows: AB-8 macroporous adsorption resin: Weigh 50 g of resin into a 500 mL beaker, add 150 mL of 4% sodium hydroxide solution, soak for 24 hours, wash with deionized water until the pH value is neutral, then wash with 150 mL of 95% ethanol solution, and then wash away the ethanol with deionized water until there is no ethanol smell in the resin, then it can be put into use.

[0029] 724 macroporous weakly acidic acrylic cation exchange resin, D101 macroporous adsorption resin, D113 macroporous weakly acidic cation exchange resin, and H103 macroporous adsorption resin: Weigh 50 g of resin and place it in a 500 mL beaker. Add 150 mL of saturated sodium chloride solution and soak for 20 hours. Drain the sodium chloride solution and rinse thoroughly with deionized water. Then soak in 150 mL of 4% sodium hydroxide solution for 3 hours. Drain the alkali solution and rinse the resin with deionized water until the pH value is neutral. Finally, soak in 150 mL of 5% hydrochloric acid for 6 hours. Drain the acid solution and rinse the resin with deionized water until the pH value is neutral. It is then ready for use.

[0030] D301 Weakly Basic Styrene-Based Anion Exchange Resin: Weigh 50 g of resin into a 500 mL beaker, add 150 mL of saturated sodium chloride solution, soak for 20 hours, then drain the sodium chloride solution, rinse thoroughly with deionized water, then soak in 150 mL of 5% hydrochloric acid for 3 hours, drain the acid, rinse the resin with deionized water until the pH is neutral, finally soak in 150 mL of 4% sodium hydroxide solution for 6 hours, drain the alkali solution, rinse the resin with deionized water until the pH is neutral, and it is ready for use.

[0031] DA201 macroporous adsorption resin, HPD-826 macroporous adsorption resin, NKA-9 macroporous adsorption resin, XAD-1180N macroporous adsorption resin: Weigh 50 g of resin and place it in a 500 mL beaker. Soak it in 150 mL of 95% ethanol solution for 6 hours. Then wash it with deionized water to remove the ethanol until there is no obvious ethanol odor in the resin. It is then ready for use.

[0032] 1.1.2 Preparation of resin-immobilized phospholipase D Weigh 1.0 g of pretreated resin into an Erlenmeyer flask, add a certain amount of crude enzyme solution of free phospholipase D-Sp-256-A409C (prepared according to the method described in CN 118895264 A), and an equal volume of 20 mmol / L pH 6.0 citrate-sodium citrate buffer to make the enzyme loading ratio (the ratio of the weight of protein in the enzyme solution to the weight of the resin) 60 mg / g. Shake and adsorb for 8 h in a constant temperature water bath at 4℃ and 220 r / min. Filter off the supernatant, and then wash repeatedly with the appropriate buffer until the supernatant does not turn blue when tested with Coomassie Brilliant Blue, thus obtaining the immobilized enzyme. Then, freeze-dry the washed and filtered immobilized enzyme for 24 h. Finally, store the dried immobilized enzyme in a sealed blue-capped bottle at 4℃.

[0033] 1.1.3 Determination of protein adsorption capacity The protein content in the crude enzyme solution and its supernatant after adsorption was determined using the Coomassie Brilliant Blue (Bradford) method.

[0034] The formula for calculating the protein adsorption capacity of immobilized enzymes is as follows: Protein adsorption capacity (mg / g) = In the formula: A This indicates the protein content (mg) in the crude enzyme solution. B This indicates the remaining protein content (mg) in the supernatant. C This indicates the mass of the carrier (resin) added.

[0035] 1.1.4 Determination of enzyme activity recovery rate The hydrolytic activities of the immobilized enzyme and the added free enzyme were determined using the following method: (1) Preparation of the standard curve for color development: Weigh 0.6981 g of choline chloride and dissolve it in 10 mL of deionized water to prepare a 0.5 mol / L choline chloride stock solution; dilute with deionized water to obtain choline chloride solutions of different concentration gradients, and develop the solution in a water bath at 37℃ for 3 h according to the hydrolysis activity determination procedure. Measure the absorbance at 500 nm and use OD as the standard. 500 A standard curve for the colorimetric solution is prepared with α as the x-axis and the actual concentration of choline chloride as the y-axis.

[0036] (2) Determination of hydrolytic activity: The hydrolytic activity of free enzyme and immobilized enzyme was determined by enzyme-linked colorimetric method. The hydrolytic activity determination system was as follows: hydrolysis reaction substrate (10 mg / mL phosphatidylcholine, solvent: ether:water = 1:9, volume ratio), 0.1 mL; 0.1 mol / L pH 6.0 citrate buffer, 10.0 μL; 0.1 mol / L pH 6.0 calcium chloride solution, 5.0 μL; 7.5% Triton X-100, 15.0 μL; enzyme solution, 100 μL. The determination conditions were as follows: after reacting in a 37℃ water bath for 20 min, the reaction was terminated by placing it in a boiling water bath for 5 min and cooling to room temperature; 0.2 mL of colorimetric solution (100 U peroxidase, 50 U choline oxidase, 100 mg 4-aminoantipyrrolidone, 50 mg phenol, 1.0 g Triton X-100) was added, and the mixture was placed in a 37℃ water bath for 3 h. After the reaction was completed, the mixture was centrifuged at 12000 r / min for 2 min, and 200 μL of the supernatant was taken to measure the absorbance at 500 nm.

[0037] Hydrolytic activity is defined as the amount of enzyme required to release 1 μmol of choline per minute under the given reaction conditions; one enzyme activity unit is defined as such.

[0038] (3) Calculate the enzyme activity recovery rate according to the following formula: Enzyme activity recovery rate (%) = In the formula, X This indicates the total enzyme activity after the addition of free enzymes. Y This indicates the total enzyme activity of the remaining free enzyme after immobilization. Z This indicates the total enzyme activity of the immobilized enzyme.

[0039] 1.1.5 Determination of the transesterification activity of immobilized enzymes DHA-PE was synthesized by transesterification using a classic biphasic reaction system with DHA-phosphatidylcholine (DHA-PC) and ethanolamine hydrochloride as substrates.

[0040] Organic phase: DHA-PC was dissolved in cyclopentyl methyl ether as an organic solvent to achieve a substrate concentration of 10 mg / mL.

[0041] Aqueous phase: 50 mM anhydrous calcium chloride was dissolved in 20 mmol / L citrate-sodium citrate buffer solution at pH 6.0, ethanolamine hydrochloride with a substrate molar ratio (ethanolamine hydrochloride: DHA-PC) of 20:1 was added, and 100 mg of resin-immobilized PLD was added.

[0042] Take 1 mL of aqueous phase and 1 mL of organic phase into a brown reaction flask, mix and shake in a water bath shaker at 40℃ and 220 r / min, and after reacting for 8 h, take it out and transfer the reaction system to a 2 mL EP tube. Centrifuge at 12000 r / min for 2 min and collect the upper layer of reaction solution.

[0043] The products were determined using high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), and the transesterification activity (i.e., substrate conversion rate of DHA-PC) was calculated. The highest transesterification activity among the 10 resin-immobilized phosphatase D was defined as 100%, and the relative transesterification activity of each resin-immobilized phosphatase D was calculated.

[0044] 1.1.6 Optimization of the Immobilization Process 1.1.6.1 Effect of immobilization time on the immobilization process Accurately weigh 0.1 g of pretreated D301 macroporous resin and place it in a 2 mL EP tube. Add a certain amount of crude enzyme solution and an equal volume of 20 mmol / L pH 6.0 citrate-sodium citrate buffer to achieve an enzyme loading ratio of 60 mg / g. The tube is then shaken and adsorbed in a constant temperature water bath at 4℃ and 220 r / min for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Filter off the supernatant and wash repeatedly with the appropriate buffer until the supernatant does not turn blue when tested with Coomassie Brilliant Blue, thus obtaining the immobilized enzyme. The protein adsorption capacity, enzyme activity recovery rate, and relative transesterification activity of the immobilized enzyme at different adsorption times are measured.

[0045] 1.1.6.2 Effect of pH on the immobilization process Accurately weigh 0.1 g of pretreated D301 macroporous resin and place it in a 2 mL EP tube. Add a certain amount of crude enzyme solution and an equal volume of 20 mmol / L citrate-sodium citrate buffer (pH set to 3.0–9.0) to achieve an enzyme loading ratio of 60 mg / g. Incubate at 4℃ and 220 r / min in a constant temperature water bath for 3 h. Filter off the supernatant and wash repeatedly with the appropriate buffer until the supernatant does not turn blue when tested with Coomassie Brilliant Blue, thus obtaining the immobilized enzyme. Measure the protein adsorption capacity, hydrolytic enzyme activity recovery rate, and relative transesterification activity of the immobilized enzyme at different pH values.

[0046] 1.1.6.3 Effect of enzyme to carrier mass ratio on immobilization process Accurately weigh 0.1 g of pretreated D301 macroporous resin and place it in a 2 mL EP tube. Add a certain amount of crude enzyme solution and an equal volume of 20 mmol / L pH 5.0 citrate-sodium citrate buffer. Set enzyme loading ratios of 10 mg / g, 20 mg / g, 40 mg / g, 60 mg / g, 80 mg / g, 100 mg / g, and 120 mg / g. Shake and adsorb for 3 h at 4℃ and 220 r / min in a constant temperature water bath. Filter off the supernatant and wash repeatedly with the corresponding buffer until the supernatant does not turn blue when tested with Coomassie Brilliant Blue, thus obtaining the immobilized enzyme. Determine the protein adsorption capacity, hydrolytic enzyme activity recovery rate, and relative transesterification activity of the immobilized enzyme at different enzyme loading ratios.

[0047] 1.1.6.4 Preparation of immobilized enzymes under optimal conditions Accurately weigh 0.1 g of pretreated D301 macroporous resin and place it in a 2 mL EP tube. Add a certain amount of crude enzyme solution and an equal volume of 20 mmol / L pH 5.0 citrate-sodium citrate buffer to make the enzyme loading ratio 60 mg / g. Shake and adsorb in a constant temperature water bath at 4℃ and 220 r / min for 3 h. Filter off the supernatant and then wash repeatedly with the appropriate buffer until the supernatant does not turn blue when tested with Coomassie Brilliant Blue. Freeze-dry to obtain the immobilized enzyme.

[0048] 1.1.7 Characterization of immobilized enzymes 1.1.7.1 Scanning Electron Microscopy (SEM) Observation The characterization changes of the material before and after immobilization were observed using scanning electron microscopy: D301 macroporous resin and immobilized enzyme (i.e., the immobilized enzyme prepared in 1.1.6.4 above, the same below) were dried in an oven at 40℃ for 8 h. The dried sample was then evenly dispersed on a conductive adhesive, and a layer of platinum was coated on the sample surface. The scanning voltage was set to 5.0 kV, and the magnification was 5 × 10⁻⁶. 4 The sample is scanned.

[0049] 1.1.7.2 Fourier Transform Infrared Spectroscopy (FT-IR) Detection The structures of D301 macroporous resin, immobilized enzyme, and free enzyme were analyzed using FT-IR to examine structural changes before and after immobilization. The resin and immobilized enzyme were dried in a 40℃ oven for 8 h. A crude enzyme solution containing phospholipase D-Sp-256-A409C was freeze-dried for 48 h to obtain crude enzyme powder of the free enzyme. The dried resin, immobilized enzyme, and free enzyme were then mixed with potassium bromide at a ratio of 1:100 (w / w). A suitable amount of the mixed sample was ground into powder in an agate mortar and pressed into a flat, uniform, and crack-free sheet in a mold. The powder was then analyzed using FT-IR at wavelengths of 400–4000 cm⁻¹. -1 A full-band infrared scan was performed, with a scan resolution set to 2 cm. -1 , 128 scans.

[0050] 1.1.8 Optimization of transesterification reaction conditions for immobilized enzyme The transesterification reaction conditions of the immobilized enzyme were optimized, and the free enzyme was used as a control group to explore the difference in transesterification reaction effects between the immobilized enzyme and the free enzyme.

[0051] 1.1.8.1 Optimization of substrate molar ratio In the transesterification reaction process (i.e., the biphasic reaction system in 1.1.5 above), the organic phase had a DHA-PC concentration of 10 mg / mL; the aqueous phase consisted of 50 mM anhydrous calcium chloride dissolved in 20 mmol / L citrate-sodium citrate buffer solution at pH 6.0, with 0.2 U (150 mg) of immobilized enzyme added and 0.2 U of free enzyme added as a control. The substrate molar ratio (ethanolamine hydrochloride:DHA-PC) was set to 5:1, 10:1, 20:1, 50:1, 100:1, and 200:1. The volume ratio of the two phases was 1:1, the reaction temperature was 40℃, and the reaction time was 8 h. After the reaction, HPLC analysis was performed to calculate the substrate conversion rate of DHA-PC. The highest transesterification activity was defined as 100%, and the relative transesterification activity was calculated.

[0052] 1.1.8.2 pH Optimization In the transesterification reaction, the organic phase had a DHA-PC concentration of 10 mg / mL; the aqueous phase consisted of 50 mM anhydrous calcium chloride dissolved in 20 mmol / L citrate-sodium citrate buffer solution (pH range 3.0–10.0), with 0.2 U (150 mg) of immobilized enzyme and 0.2 U of free enzyme added as a control, and a substrate molar ratio of 10:1. The volume ratio of the two phases was 1:1, the reaction temperature was 40℃, and the reaction time was 8 h. After the reaction, HPLC analysis was performed to calculate the substrate conversion rate of DHA-PC, defining the highest transesterification activity as 100%, and calculating the relative transesterification activity.

[0053] 1.1.8.3 Temperature Optimization In the transesterification reaction, the organic phase consisted of DHA-PC at a concentration of 10 mg / mL; the aqueous phase consisted of 50 mM anhydrous calcium chloride dissolved in 20 mmol / L citrate-sodium citrate buffer solution at pH 5.0, with 0.2 U (150 mg) of immobilized enzyme added and 0.2 U of free enzyme added as a control, and a substrate molar ratio of 10:1. The volume ratio of the two phases was 1:1, and the reaction temperatures were set at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, with a reaction time of 8 h. After the reaction, HPLC analysis was performed to calculate the substrate conversion rate of DHA-PC, defining the highest transesterification activity as 100%, and calculating the relative transesterification activity.

[0054] 1.1.8.4 Optimization of the organic phase / water ratio In the transesterification reaction, the organic phase concentration of DHA-PC was 10 mg / mL; the aqueous phase consisted of 50 mM anhydrous calcium chloride dissolved in 20 mmol / L citrate-sodium citrate buffer solution at pH 5.0, with 0.2 U (150 mg) of immobilized enzyme added and 0.2 U of free enzyme added as a control. The substrate molar ratio was set to 10:1. Organic phase / water ratios were set at 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4, respectively. The reaction temperature was 60℃, and the reaction time was 8 h. After the reaction, HPLC analysis was performed to calculate the substrate conversion rate of DHA-PC. The highest transesterification activity was defined as 100%, and the relative transesterification activity was calculated.

[0055] 1.1.9 Operational stability of resin-immobilized phospholipase D Weigh 0.1 g of immobilized enzyme and determine its hydrolytic activity according to the reaction system described in 1.1.4 above. After the reaction lasts for 20 min, remove all liquid from the EP tube and inactivate it by boiling in a water bath for 5 min. Wash the remaining immobilized enzyme in the EP tube with buffer and repeat the reaction with the same system for the next batch. Then, continue to take samples for inactivation and repeat the operation. Determine the hydrolytic activity of each batch using an enzyme-linked colorimetric method. Define the hydrolytic activity of the first batch as 100% and calculate the relative enzyme activity of different batches to investigate the operational stability of the resin-immobilized PLD.

[0056] 1.2 Results and Discussion 1.2.1 Screening of Resin Immobilization Carriers In this experiment, ten macroporous resins, namely DA201, HPD-826, NKA-9, AB-8, XAD 1180N, 724, D101, D113, H103 and D301, were selected as immobilized enzyme carriers. The physical properties of these resins are compared in Table 1, which covers polar, weakly polar, non-polar, adsorption resins, cation exchange resins and anion exchange resins.

[0057]

[0058] The results of determining the protein adsorption capacity, enzyme activity recovery rate, and relative transesterification activity of 10 resin-immobilized enzymes are as follows: Figure 1 As shown in the figure, among the 10 selected macroporous resins, the D301 weakly basic styrene-based anion exchange resin used as a carrier exhibited the highest transesterification activity for the immobilized enzyme. This may be because D301, as a weakly basic anion exchange resin, is rich in positively charged tertiary amine groups [-N(CH3)] on its surface. 2 Through electrostatic attraction, enzymes and carriers form stable ionic bonds, reducing enzyme molecule shedding. Next are D101 macroporous adsorption resin and NKA-9 macroporous adsorption resin, while 724 macroporous weakly acidic acrylic cation exchange resin and D113 macroporous weakly acidic cation exchange resin exhibit the worst transesterification activity, with almost no transesterification activity. In terms of protein adsorption capacity and enzyme activity recovery rate, the 724 macroporous weakly acidic acrylic cationic macroporous adsorption resin had the highest protein adsorption capacity at 17.22 mg / g, but its enzyme activity recovery rate was only 27.3%. The NKA-9 macroporous adsorption resin was second, but its enzyme activity recovery rate was also very low at only 25.2%. The resins with the lowest protein adsorption capacity were AB-8, D101, and D113, with adsorption capacities of 5.06 mg / g, 5.32 mg / g, and 5.44 mg / g, respectively. However, AB-8 and D101 had very high enzyme activity recovery rates, reaching 77.7% and 94.5%, respectively. The remaining resins had protein adsorption capacities ranging from 8 to 12 mg / g, and except for D301, the enzyme activity recovery rates of the other macroporous resins were all below 50%. Overall, with the same immobilization material and enzyme solution consumption, D301 exhibited the highest transesterification activity and better met the screening criteria. Therefore, D301 weakly basic styrene-based anion exchange resin was chosen as the immobilization carrier for further investigation.

[0059] 1.2.2 Optimization of the immobilization process 1.2.2.1 Effect of immobilization time on the immobilization process The results of measuring the protein adsorption capacity, enzyme activity recovery rate, and relative transesterification activity of the immobilized enzyme at different adsorption times are as follows: Figure 2As shown in the figure, the protein adsorption capacity gradually increased within the first 4 hours of immobilization, but approached saturation after 4 hours as the adsorption time increased. The relative transesterification activity and enzyme activity recovery rate reached their highest values ​​when the immobilization time was 3 hours, with an enzyme activity recovery rate of 89.5%. When the immobilization time increased to 4–6 hours, the transesterification activity remained almost unchanged, but the enzyme activity recovery rate decreased slightly. This may be because when the adsorption time was too long, the amount of enzyme bound to the carrier approached saturation, causing the pores to be blocked by too many PLD enzyme molecules, hindering the binding of the enzyme to the substrate. Therefore, an adsorption time of 3 hours is the optimal adsorption time.

[0060] 1.2.2.2 Effect of buffer pH on the immobilization process The results of measuring the protein adsorption capacity, hydrolytic enzyme activity recovery rate, and relative transesterification activity of the immobilized enzyme at different pH values ​​are as follows: Figure 3 As shown in the figure, different pH values ​​did not significantly affect the protein adsorption capacity. When the buffer pH increased from 3.0 to 9.0, the transesterification activity of the immobilized PLD showed a trend of first increasing and then decreasing, and the overall trend of enzyme activity recovery was consistent with the overall trend of transesterification activity. When the buffer pH was 5.0, the relative transesterification activity and hydrolytic enzyme activity recovery rate of the immobilized enzyme were the highest, with an enzyme activity recovery rate of 95.1%. The transesterification activity of the immobilized enzyme showed a trend of first increasing and then decreasing because, on the one hand, changes in pH environment can alter the surface charge distribution of the carrier and enzyme molecules, and may also change the interaction forces between the carrier and enzyme molecules; on the other hand, enzyme stability is poor when the pH is acidic or alkaline, leading to a decrease in the activity of the immobilized enzyme. Therefore, a buffer pH of 5.0 is the optimal pH.

[0061] 1.2.2.3 Effect of enzyme to carrier mass ratio on immobilization process The results of measuring the protein adsorption capacity, hydrolytic enzyme activity recovery rate, and relative transesterification activity of immobilized enzymes at different enzyme loading ratios are as follows: Figure 4 As shown in the figure, the protein adsorption gradually increases with increasing enzyme loading ratio, reaching a maximum of 20.1 mg / g when the enzyme loading ratio is 120 mg / g. With increasing enzyme loading ratio, both transesterification activity and enzyme activity recovery rate show a trend of first increasing and then decreasing, reaching their highest values ​​at an enzyme loading ratio of 60 mg / g, with an enzyme activity recovery rate of 94.5%. The enzyme activity initially increases with increasing enzyme loading ratio because more enzyme molecules can bind to the substrate as the protein adsorption increases, leading to a corresponding increase in activity. However, when the enzyme loading ratio exceeds 60 mg / g, it may disrupt the monolayer distribution of PLD molecules in the resin channels, causing blockage and preventing sufficient exposure of the enzyme's active site, hindering substrate binding and reducing enzyme activity. Therefore, an enzyme loading ratio of 60 mg / g is the optimal enzyme loading ratio.

[0062] 1.2.3 Characterization of immobilized enzymes 1.2.3.1 SEM Test Analysis The surface morphology of unimmobilized resin material and resin-immobilized PLD was observed using scanning electron microscopy, such as... Figure 5 As shown in the figure, the surface of the unimmobilized D301 macroporous resin material has a large number of cavities. These gaps provide adsorption space for phospholipase D molecules and also provide a larger contact area during the reaction, which is more conducive to the binding of substrate and enzyme molecules and improves the product yield. The surface of the immobilized D301 macroporous resin pores is covered by enzyme molecules, and the pores are smaller, indicating that PLD molecules are effectively bound to the D301 macroporous resin. Therefore, it is believed that this immobilization method can successfully prepare resin-immobilized phospholipase D through physical adsorption.

[0063] 1.2.3.2 FT-IR Test Analysis The immobilized enzyme was further characterized and validated using Fourier transform infrared spectroscopy, and the results are as follows: Figure 6 As shown. In the infrared spectrum of D301 macroporous resin and immobilized enzyme, 3100–3000 cm⁻¹ -1 Represents the stretching vibration of the unsaturated carbon-hydrogen bond (=CH) of the benzene ring, 811 cm⁻¹ -1 The absorption peaks at these locations represent the vibrations of substituents on the benzene ring; these two absorption peaks are absent in the infrared spectrum of the free enzyme. In the infrared spectrum of the immobilized enzyme, there are no very obvious characteristic peaks of the free enzyme, which may be due to the much lower enzyme molecule content in the immobilized enzyme compared to the resin material. However, the 3500–3100 cm⁻¹ peaks... -1 Represents the stretching vibration of the NH bond, 1680–1500 cm⁻¹ -1 The stretching vibrations of the peptide bond -CONH, and the shifts in the -NH and -CONH peaks, also demonstrate the presence of the enzyme. Experimental verification confirmed the immobilized enzyme's activity, confirming the successful immobilization of phospholipase D-Sp-256-A409C on the macroporous resin. Furthermore, the absence of new major peaks in the infrared spectrum, other than those related to the material and the enzyme, indicates that phospholipase D-Sp-256-A409C is immobilized on the D301 macroporous resin through non-covalent interactions (i.e., physical adsorption).

[0064] 1.2.4 Optimization of immobilized enzyme transesterification reaction 1.2.4.1 Optimal substrate molar ratio The optimal substrate molar ratio for transesterification of the immobilized enzyme was investigated, while the differences in transesterification characteristics between the immobilized and free enzymes were explored using a free enzyme of the same activity as a control. The results are as follows: Figure 7As shown, both immobilized and free enzymes exhibit a trend of first increasing and then decreasing with the increase of substrate molar ratio. This is because when the concentration of ethanolamine hydrochloride in the reaction system is low, some PLDs are still in an idle state. As the concentration increases, ethanolamine hydrochloride can bind to PLDs and participate in the reaction quickly, thus increasing the transesterification activity. However, when the concentration of ethanolamine hydrochloride reaches a certain level, all PLD molecules are in a bound state. Even if the concentration of ethanolamine hydrochloride is increased, the reaction rate cannot be increased. Furthermore, excessively high substrate concentrations can cause steric hindrance, which can actually reduce transesterification efficiency.

[0065] The results show that the optimal substrate molar ratio for resin-immobilized PLDs is 10:1, while that for free PLDs is 20:1. The lower optimal substrate molar ratio for immobilized enzymes compared to free enzymes is due to the difference in the number of enzyme molecules between immobilized and free PLDs with the same hydrolytic activity; the immobilized enzyme contains fewer enzyme molecules than the free enzyme. For a given enzyme activity, immobilized enzymes can produce more DHA-PE using less ethanolamine hydrochloride, which is more cost-effective and beneficial for industrial production.

[0066] 1.2.4.2 Optimal pH The optimal pH for resin-immobilized PLDs to participate in the transesterification reaction was investigated. Results are as follows: Figure 8 As shown, the immobilized enzyme exhibits excellent stability within a pH range of 3.0–10.0, maintaining good transesterification activity under both acidic and alkaline conditions. In contrast, the free enzyme is highly sensitive, with transesterification efficiency varying significantly with pH changes. The free PLD shows optimal transesterification activity at pH 5.0, while its activity decreases significantly at pH values ​​below 5.0 or above 9.0, indicating protein denaturation under excessively acidic or alkaline conditions. This demonstrates that resin-immobilized PLDs offer a wider pH range, greater operability, and are more suitable for industrial production compared to free enzymes.

[0067] 1.2.4.3 Optimal Temperature The effect of temperature on the transesterification reaction of resin-immobilized PLD was investigated. The results are as follows: Figure 9As shown, the optimal temperature for free enzymes is 40℃, while the optimal temperature for resin-immobilized enzymes is 60℃. Except for 30℃ and 80℃, the relative enzyme activity of resin-immobilized PLDs is higher than that of free PLDs at other temperature conditions. This may be because when the temperature is below 40℃, the energy supply in the system is insufficient, which is not conducive to the substrate entering the resin pores and binding to the enzyme molecules; when the temperature reaches 80℃, the enzyme denatures and becomes inactive, almost losing its transesterification activity; while in the range of 40℃ to 70℃, D301 resin provides a more stable microenvironment for phospholipase D, playing a protective role for the enzyme protein, giving it a higher activation energy, and improving its relative activity. Therefore, the optimal temperature for resin-immobilized PLDs is higher than that for free enzyme PLDs.

[0068] 1.2.4.4 Optimal organic phase / water ratio The effect of the organic phase / water ratio on the transesterification reactions of immobilized and free enzymes was investigated. The results are as follows: Figure 10 As shown, the optimal organic phase / water ratio for the free enzyme is 1:1, while that for the immobilized enzyme is 1:2. The difference in the optimal organic phase / water ratio between the resin-immobilized PLD and the free PLD may be due to the hygroscopic nature of the resin material; therefore, a higher water content is more conducive to the transesterification reaction of the immobilized enzyme. Furthermore, the relative transesterification activity of the immobilized enzyme is consistently higher than that of the free enzyme; the highest transesterification activity of the free enzyme is only 63.2% of the highest activity of the immobilized enzyme, indicating that the activity of phospholipase D is indeed improved after immobilization.

[0069] Based on the optimization results, the optimal transesterification reaction conditions are as follows: Organic phase: DHA-PC concentration of 10 mg / mL; Aqueous phase: 50 mM anhydrous calcium chloride dissolved in 20 mmol / L citrate-sodium citrate buffer solution (pH 3.0–10.0), with 0.2 U (150 mg) of immobilized enzyme added, substrate molar ratio of 10:1; Organic phase / water ratio of 1:2; Reaction temperature of 60℃; Reaction time of 8 h.

[0070] 1.2.5 Operational stability of resin-immobilized PLD One major advantage of immobilized enzymes compared to free enzymes is their reusability. Therefore, the reusability of immobilized enzymes was investigated by measuring their hydrolytic activity. The results are as follows: Figure 11 As shown, after three reuses, the residual enzyme activity of resin-immobilized PLD still reached over 90%, and after seven reuses, it still reached 84.6%. In previous studies, PLD immobilized on magnetic Fe3O4 / SiO2 nanomaterials retained over 40% of its hydrolytic enzyme activity after eight reuses. The comparison demonstrates that resin-immobilized PLD has significant advantages over free PLD.

[0071] 1.3 Conclusion This experiment used a physical adsorption method to immobilize phospholipase D-Sp-256-A409C, and the conclusions are as follows: (1) Based on the transesterification activity, hydrolysis activity recovery rate and protein adsorption amount, D301 weakly basic styrene-based anion exchange resin was selected as the immobilization carrier from 10 macroporous resins.

[0072] (2) Optimize the enzyme immobilization process: The optimal immobilization conditions are 3 h immobilization time, 20 mmol / L pH 5.0 citrate-sodium citrate buffer solution, and enzyme loading ratio of 60 mg / g.

[0073] (3) The immobilized enzyme was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy, which confirmed that the free PLD was immobilized on D301 weakly basic styrene-based anion exchange resin through non-covalent interactions.

[0074] (4) The application of resin-immobilized phospholipase D in the synthesis of DHA-PE was systematically studied and compared with free enzymes of the same hydrolytic activity. It was found that the immobilized enzyme has a wider applicable temperature and pH range than the free enzyme, and its stability and activity are improved. The optimal substrate molar ratio of the resin-immobilized PLD transesterification system is 10:1. The transesterification activity remains stable in the pH range of 3.0 to 10.0. The optimal temperature is 60℃, and the optimal organic phase / water ratio of the immobilized enzyme is 1:2.

[0075] (5) Its operational stability was studied. After the resin-immobilized PLD was reused 3 times, its residual enzyme activity was still more than 90%. After being reused 7 times, the residual enzyme activity of the immobilized enzyme was still 84.6%.

[0076] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An immobilized phospholipase D, characterized in that, It was prepared by the following method: phospholipase D-Sp-256-A409C was loaded onto D301 weakly basic styrene-based anion exchange resin at an enzyme loading ratio of 10-120 mg / g; the amino acid sequence of the phospholipase D-Sp-256-A409C is shown in SEQ ID NO.

1.

2. The immobilized phospholipase D according to claim 1, characterized in that: The enzyme loading ratio is 60 mg / g.

3. The immobilized phospholipase D according to claim 1, characterized in that, The enzyme was prepared by adding an enzyme solution containing phospholipase D-Sp-256-A409C and a citrate-sodium citrate buffer solution with a pH of 3.0–10.0 to a D301 weakly basic styrene-based anion exchange resin to make the enzyme loading ratio 10–120 mg / g; shaking adsorption for 1–6 h; filtering off the supernatant, rinsing with buffer solution, and freeze-drying to obtain the immobilized enzyme.

4. The immobilized phospholipase D according to claim 3, characterized in that, The enzyme was prepared by the following method: an enzyme solution containing phospholipase D-Sp-256-A409C was added to a D301 weakly basic styrene-based anion exchange resin, along with an equal volume of 20 mmol / L pH 5.0 citrate-sodium citrate buffer to achieve an enzyme loading ratio of 60 mg / g; the enzyme was adsorbed by shaking at 4℃ and 220 r / min for 3 h; the supernatant was filtered off, the enzyme was washed with buffer, and then freeze-dried to obtain the immobilized enzyme.

5. The immobilized phospholipase D according to claim 3 or 4, characterized in that, The D301 weakly basic styrene-based anion exchange resin was pretreated as follows: 50 g of D301 weakly basic styrene-based anion exchange resin was taken and 150 mL of saturated sodium chloride solution was added. The resin was soaked for 18–20 hours. The sodium chloride solution was drained, and the resin was rinsed with deionized water. The resin was then soaked in 150 mL of 5% hydrochloric acid for 2–4 hours. The acid solution was drained, and the resin was rinsed with deionized water until the pH value was neutral. The resin was then soaked in 150 mL of 4% sodium hydroxide solution for 4–8 hours. The alkali solution was drained, and the resin was rinsed with deionized water until the pH value was neutral.

6. The use of immobilized phospholipase D according to any one of claims 1 to 5 in the preparation of DHA-PE.

7. The application according to claim 6, characterized in that: In practical applications, DHA-phosphatidylcholine and ethanolamine hydrochloride are used as substrates, and DHA-PE is synthesized through transesterification under the catalysis of immobilized phospholipase D.

8. The application according to claim 7, characterized in that, The method for preparing DHA-PE is as follows: an organic phase and an aqueous phase are mixed and reacted at 30–70 °C; the aqueous phase is a 10 mg / mL DHA-PC solution, and the solvent is cyclopentyl methyl ether; the organic phase is prepared by dissolving 50 mM anhydrous calcium chloride in a 20 mmol / L citrate-sodium citrate buffer solution with a pH of 3.0–10.0, and adding 150 mg of immobilized phospholipase D; the molar ratio of ethanolamine hydrochloride to DHA-PC is (5–200):1; the volume ratio of the organic phase to the aqueous phase is (1–3):(1–4).

9. The application according to claim 8, characterized in that: The reaction temperature is 40°C; the molar ratio of ethanolamine hydrochloride to DHA-PC is 10:1; and the volume ratio of the organic phase to the aqueous phase is 1:2.

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